Mesenchymal stromal cells and uses related thereto

ABSTRACT

The present invention generally relates to novel preparations of mesenchymal stromal cells (MSCs) derived from hemangioblasts, methods for obtaining such MSCs, and methods of treating a pathology using such MSCs. The methods of the present invention produce substantial numbers of MSCs having a potency-retaining youthful phenotype, which are useful in the treatment of pathologies.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 13/905,526, filedMay 30, 2013, now allowed, which is a continuation-in-part of U.S. Ser.No. 13/691,349, filed Nov. 30, 2012, now allowed, which claims thebenefit of priority to U.S. Ser. No. 61/565,358, filed Nov. 30, 2011,entitled “METHODS OF GENERATING MESENCHYMAL STROMAL CELLS USINGHEMANGIOBLASTS”, each of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the use of cell-based therapies toreduce the manifestations of a pathology such as that characterized byan inappropriate immune response in a subject, and also to affect theorigin of a pathology such that the abnormality defining the pathologyis returned to a normal posture. In particular, the present inventionrelates to mesenchymal stromal cells (MSCs) that retain a phenotype of“youthful” cells that imparts a high potency in the reduction of amanifestation of a pathology in a subject.

BACKGROUND OF THE INVENTION

Many pathologies manifest clinically through unwanted or excessiveimmune responses within a host, e.g., transplant rejection, inflammatoryand autoimmune disorders. Immunosuppressive therapies have beendeveloped to treat the symptoms, but not the underlying cause ofpathologies characterized by excessive immune responses. These therapiesare effective at down-modulating immune function and, as such, carry thepotential for severe adverse events, including cancer and opportunisticinfection, as well as side effects such as cataracts, hyperglycemia,bruising, and nephrotoxicity from agents such as prednisone,cyclosporine, and tacrolimus.

Although therapies that do not suppress the entire immune system havebeen developed, there are limitations associated with these regimens aswell. These immunomodulatory treatments target a narrower point ofintervention within the immune system and, as such, have different,sometimes less severe side effects. Examples of such immunomodulatorytherapies include the use of antibodies, e.g., anti-CD3 or anti-IL2R.While successful at inducing a heightened state of non-responsiveness,the withdrawal of these immunomodulatory therapies results in areversion to the unwanted pathology.

Mesenchymal stem cells (MSC or MSCs) are multipotent stem cells withself-renewal capacity and the ability to differentiate into osteoblasts,chondrocytes, and adipocytes, among other mesenchymal cell lineages. Inrecent years, the intense research on the multilineage differentiationpotential and immunomodulatory properties of human MSC has indicatedthat these cells can be used to treat a range of clinical conditions,including immunological disorders as well as degenerative diseases.Consequently, the number of clinical studies with MSC has been steadilyincreasing for a wide variety of conditions: graft-versus-host disease(GVHD), myocardial infarction and inflammatory and autoimmune diseasesand disorders, among others. Currently, clinical programs utilizing MSCsrely on isolation of these cells from adult sources and cord blood. Thehigh cell doses required for MSC clinical applications (up to severalmillion cells per kg of the patient) demands a reliable, reproducibleand efficient expansion protocol, capable of generating a large numberof cells from those isolated from the donor source.

To reach the clinically meaningful cell numbers for cellular therapy andtissue engineering applications, MSC ex-vivo expansion is mandatory.Sequential ex-vivo cell passaging of MSCs from cord blood, fetal andadult sources (such as bone marrow or adipose tissues) can causereplicative stress, chromosomal abnormalities, or other stochasticcellular defects, resulting in the progressive loss of theproliferative, clonogenic and differentiation potential of the expandedMSCs, which ultimately can jeopardize MSC clinical safety and efficacy.The issues with use of senescent MSCs in treatment should not beunderestimated since cells lose part of their differentiation potentialand their secretory profile is also altered. MSC senescence duringculture was found to induce cell growth arrest, concurrently withtelomere shortening. A continuous decrease in adipogenic differentiationpotential was reported for bone marrow (BM) MSC along increasingpassages, whereas the propensity for differentiation into the osteogeniclineage increased.

Accordingly, some essential problems remain to be solved before theclinical application of MSC. MSCs derived from ESCs can be generated insufficient quantities and in a highly controllable manner, thusalleviating the problems with donor-dependent sources. Since long-termengraftment of MSCs is not required, there is basically no concern formismatch of major histocompatibility (MHC) [7, 8]. In the art, MSCsderived from ESCs have been obtained through various methods includingco-culture with murine OP9 cells or handpicking procedures [9-13]. Thesemethods, however, are tedious and generate MSCs with a low yield,varying quality and a lack of potency. Moreover, maximizing the potencyof the injected cells is desirable, in terms of being able to provide acellular product with a better therapeutic index, ability to be used ata reduce dosage (number of cells) relative to CB-derived, BM-derived oradipose-derived MSCs, and/or the ability for the MSCs to provide atractable therapy for inflammatory and autoimmune diseases for whichCB-derived, BM-derived or adipose-derived MSCs are not efficaciousenough.

SUMMARY OF PREFERRED EMBODIMENTS

The present invention relates to mesenchymal stromal cells (MSCs) andmethods for generating MSCs. The methods of the present inventionproduce substantial numbers of high quality mesenchymal stromal cells,characterized by the phenotype of youthful cells that imparts a highpotency. In an embodiment of the invention, the MSCs are derived fromhemangioblasts. Preparations of the subject MSCs are useful in thetreatment of pathologies, including unwanted immune responses, e.g.,autoimmune diseases and disorders, as well as inflammatory diseases anddisorders.

In one aspect, the present invention comprises improved preparations ofMSCs generated from hemangioblasts using improved methods for culturingthe hemangioblasts. In exemplary embodiments, mesenchymal stromal cellsof the present invention retain higher levels of potency and do notclump or clump substantially less than mesenchymal stromal cells deriveddirectly from embryonic stems cells (ESCs). Mesenchymal stromal cellsgenerated according to any one or more of the processes of the presentinvention may retain higher levels of potency, and may not clump or mayclump substantially less than mesenchymal stromal cells derived directlyfrom ESCs.

In one aspect, the invention provides pharmaceutical preparationscomprising mesenchymal stromal cells, wherein said mesenchymal stromalcells are able to undergo at least 10 population doublings, e.g., atleast 10 population doublings occur within about 22-27 days. In anotheraspect, the invention provides pharmaceutical preparations comprisingmesenchymal stromal cells, wherein said mesenchymal stromal cells areable to undergo at least 15 population doublings, e.g., at least 15population doublings occur within about 22-27 days. The pharmaceuticalpreparations may be produced by in vitro differentiation ofhemangioblasts. The mesenchymal stromal cells may be primate cells,e.g., human cells. The mesenchymal stromal cells may be able to undergoat least 15 population doublings. For example, the mesenchymal stromalcells undergo at least 20, 25, 30, 35, 40, 45, 50 or more populationdoublings. The preparation may comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%,0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%,0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%,0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%,or 0.0001% pluripotent cells. Preferably, the preparation is devoid ofpluripotent cells. The preparation may comprise at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.

In one aspect, at least 50% of said mesenchymal stromal cells arepositive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1,CXCL1, CD105, CD73 and CD90; (ii) at least one of CD10, CD24, IL-11,AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44, CD166,CD274, and HLA-ABC; or (iii) any combination thereof. In another aspect,at least 50% of said mesenchymal stromal cells are positive for (i) atleast two of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 andCD90; (ii) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73,CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC. In yet anotheraspect, at least 50% of said mesenchymal stromal cells are (i) positivefor all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90,CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC and (ii) do notexpress or express low levels of at least one of CD31, 34, 45, 133,FGFR2, CD271, Stro-1, CXCR4, TLR3. Additionally, at least 60%, 70%, 80%or 90% of such mesenchymal stromal cells may be positive for (i) one ormore of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90;or (ii) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105,CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

In one aspect, the pharmaceutical preparation comprises an amount ofmesenchymal stromal cells effective to treat or prevent an unwantedimmune response in a subject in need thereof. The pharmaceuticalpreparation may further comprise other cells, tissues or organs fortransplantation into a recipient in need thereof. Exemplary other cellsor tissues include RPE cells, skin cells, corneal cells, pancreaticcells, liver cells, or cardiac cells or tissue containing any of saidcells. In another aspect, the pharmaceutical preparation comprises anamount of mesenchymal stromal cells effective to treat or prevent pain,heat sensitivity, and/or cold sensitivity.

In another aspect, the mesenchymal stromal cells are not derived frombone marrow and the potency of the preparation in an immune regulatoryassay is greater than the potency of a preparation of bone marrowderived mesenchymal stromal cells, e.g., at least 20%, 25%, 30%, 35%,40%, 45%, 50% greater than the potency of a preparation of bone marrowderived mesenchymal stromal cells. Potency may be assayed by an immuneregulatory assay that determines the EC50 dose.

In one aspect, the preparation comprising MSCs retains between about 50and 100% of its proliferative capacity after ten population doublings.

In another aspect, the mesenchymal stromal cells of the pharmaceuticalpreparation are not derived directly from pluripotent cells and saidmesenchymal stromal cells of the pharmaceutical preparation (a) do notclump or clump substantially less than mesenchymal stromal cells deriveddirectly from ESCs; (b) more easily disperse when splitting compared tomesenchymal stromal cells derived directly from ESCs; (c) are greater innumber than mesenchymal stromal cells derived directly from ESCs whenstarting with equivalent numbers of ESCs; and/or (d) acquirecharacteristic mesenchymal cell surface markers earlier than mesenchymalstromal cells derived directly from ESCs.

The present invention further encompasses methods for generatingmesenchymal stromal cells comprising culturing hemangioblast cells underconditions that give rise to mesenchymal stem cells. The hemangioblastsmay be cultured in feeder-free conditions. Additionally, hemangioblastsmay be plated on a matrix, e.g., comprising transforming growth factorbeta (TGF-beta), epidermal growth factor (EGF), insulin-like growthfactor 1, bovine fibroblast growth factor (bFGF), and/orplatelet-derived growth factor (PDGF). The matrix may be selected fromthe group consisting of: laminin, fibronectin, vitronectin,proteoglycan, entactin, collagen, collagen 1, collagen IV, heparansulfate, Matrigel (a soluble preparation from Engelbreth-Holm-Swarm(EHS) mouse sarcoma cells), a human basement membrane extract, and anycombination thereof. The matrix may comprise a soluble preparation fromEngelbreth-Holm-Swarm mouse sarcoma cells.

In one aspect, the mesenchymal stromal cells are mammalian. Preferably,the mesenchymal stromal cells are human, canine, or equine.

In one aspect, the hemangioblasts may be cultured in a medium comprisingαMEM. In another aspect, the hemangioblasts may be cultured in a mediumcomprising serum or a serum replacement. For example, the hemangioblastscells may be cultured in a medium comprising, αMEM supplemented with 0%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, or 20% fetal calf serum. In additional exemplaryembodiments the medium may comprise higher percentages of fetal calfserum, e.g., more than 20%, e.g., at least 25%, at least 30%, at least35%, at least 40%, or even higher percentages of fetal calf serum. Thehemangioblasts may be cultured on said matrix for at least about 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

In one aspect, the hemangioblasts are differentiated from pluripotentcells, e.g., iPS cells, or blastomeres. The pluripotent cells may bederived from one or more blastomeres without the destruction of a humanembryo. Additionally, the hemangioblasts may be differentiated frompluripotent cells by a method comprising (a) culturing said pluripotentcells to form clusters of cells. In one aspect, the pluripotent cellsare cultured in the presence of vascular endothelial growth factor(VEGF) and/or bone morphogenic protein 4 (BMP-4). VEGF and BMP-4 may beadded to the pluripotent cell culture within 0-48 hours of initiation ofsaid cell culture, and said VEGF is optionally added at a concentrationof 20-100 nm/mL and said BMP-4 is optionally added at a concentration of15-100 ng/mL.

In one aspect, the hemangioblasts are differentiated from pluripotentcells by a method further comprising: (b) culturing said single cells inthe presence of at least one growth factor in an amount sufficient toinduce the differentiation of said clusters of cells intohemangioblasts. The at least one growth factor added in step (b) maycomprise one or more of basic fibroblast growth factor (bFGF), vascularendothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4),stem cell factor (SCF), Flt 3L (FL), thrombopoietin (TPO), EPO, and/ortPTD-HOXB4. The one or more of said at least one growth factor added instep (b) may be added to said culture within 36-60 hours from the startof step (a). Preferably, the one or more of said at least one growthfactor added in step (b) is added to said culture within 40-48 hoursfrom the start of step (a). The at least one factor added in step (b)may comprise one or more of bFGF, VEGF, BMP-4, SCF, FL and/ortPTD-HOXB4. The concentration of said growth factors if added in step(b) may range from about the following: bFGF is is about 20-25 ng/ml,VEGF is about 20-100 ng/ml, BMP-4 is about 15-100 ng/ml, SCF is about20-50 ng/ml, FL is about 10-50 ng/ml, TPO is about 20-50 ng/ml, andtPTD-HOXB4 is about 1.5-5 U/ml.

In another aspect, the method further comprises (c) dissociating saidclusters of cells, optionally into single cells. In another aspect, themethod further comprises (d) culturing said hemangioblasts in a mediumcomprising at least one additional growth factor, wherein said at leastone additional growth factor is in an amount sufficient to expand thehemangioblasts. The at least one additional growth factor of (d) maycomprise one or more of: insulin, transferrin, granulocyte macrophagecolony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6(IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin(EPO), stem cell factor (SCF), vascular endothelial growth factor(VEGF), bone morphogenic protein 4 (BMP-4), and/or tPTD-HOXB4. Exemplaryconcentrations in step (d) include insulin about 10-100 μg/ml,transferrin about 200-2,000 μg/ml, GM-CSF about 10-50 ng/ml, IL-3 about10-20 ng/ml, IL-6 about 10-1000 ng/ml, G-CSF about 10-50 ng/ml, EPOabout 3-50 U/ml, SCF about 20-200 ng/ml, VEGF about 20-200 ng/ml, BMP-4about 15-150 ng/ml, and/or tPTD-HOXB4 about 1.5-15 U/ml. The medium instep (a), (b), (c) and/or (d) may be a serum-free medium.

In one aspect, the method generates at least 80, 85, 90, 95, 100, 125,or 150 million mesenchymal stromal cells. The hemangioblasts may beharvested after at least 10, 11, 12, 13, 14, 15, 16, 17 or 18 days ofstarting to induce differentiation of said pluripotent cells. Themesenchymal stromal cells may be generated within at least 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 days of starting to induce differentiation of saidpluripotent cells. In another aspect, the method results in at least 80,85, 90, 95, 100, 125, or 150 million mesenchymal stromal cells beinggenerated from about 200,000 hemangioblasts within about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35days of culture. The mesenchymal stromal cells may be generated fromhemangioblasts in a ratio of hemangioblasts to mesenchymal stromal cellsof at least 1:200, 1:250, 1:300, 1:350, 1:400, 1:415, 1:425, 1:440;1:450, 1:365, 1:475, 1:490 and 1:500 within about 26, 27, 28, 29, 30,31, 32, 33, 34 or 35 days of culture. The cells may be human.

The present invention also contemplates mesenchymal stromal cellsderived from hemangioblasts obtained by the described methods. In oneaspect, the invention includes mesenchymal stromal cells derived by invitro differentiation of hemangioblasts. At least 50% of saidmesenchymal stromal cells may (i) be positive for all of CD10, CD24,IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44,CD166, CD274, and HLA-ABC and (ii) not express or express low levels ofat least one of CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, TLR3.Alternatively, at least 50% of said mesenchymal stromal cells may bepositive for (i) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105,CD73 and CD90; or (ii) all of CD73, CD90, CD105, CD13, CD29, CD44,CD166, CD274, and HLA-ABC. At least 60%, 70%, 80% or 90% of thesemesenchymal stromal cells may be positive for (i) at least one of CD10,CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (ii) atleast one of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, andHLA-ABC. Preferably, the mesenchymal stromal cells do not express orexpress low levels of at least one of CD31, CD34, CD45, CD133, FGFR2,CD271, Stro-1, CXCR4, TLR3.

In another aspect, the invention encompasses a preparation of themesenchymal stromal cells described herein. The preparation may compriseless than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%,0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%,0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% pluripotent cells.Preferably, the preparation is devoid of pluripotent cells. Thepreparation may be substantially purified and optionally comprises atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% humanmesenchymal stromal cells. The preparation may comprise substantiallysimilar levels of p53 and p21 protein or the levels of p53 protein ascompared to p21 protein may be 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 timesgreater. The mesenchymal stromal cells may be capable of undergoing atleast 5 population doublings in culture. Preferably, the mesenchymalstromal cells are capable of undergoing at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55.60 or more population doublings in culture.

In one aspect, the mesenchymal stromal cells of the present invention(a) do not clump or clump substantially less than mesenchymal stromalcells derived directly from ESCs; (b) more easily disperse whensplitting compared to mesenchymal stromal cells derived directly fromESCs; (c) are greater in number than mesenchymal stromal cells deriveddirectly from ESCs when starting with equivalent numbers of ESCs; and/or(d) acquire characteristic mesenchymal cell surface markers earlier thanmesenchymal stromal cells derived directly from ESCs. The inventioncontemplates a pharmaceutical preparation comprising such mesenchymalstromal cells, which comprises an amount of mesenchymal stromal cellseffective to treat an unwanted immune response. The invention alsocontemplates a pharmaceutical preparation comprising such mesenchymalstromal cells, which comprises an amount of mesenchymal stromal cellseffective to treat or prevent pain, heat sensitivity, and/or coldsensitivity. The preparation may comprise an amount of mesenchymalstromal cells effective to treat an unwanted immune response and mayfurther comprise other cells or tissues for transplantation into arecipient in need thereof. Exemplary other cells include allogeneic orsyngeneic pancreatic, neural, liver, RPE, or conical cells or tissuescontaining any of the foregoing. The pharmaceutical preparation may beuseful in treating an autoimmune disorder or an immune reaction againstallogeneic cells including, but not limited to, multiple sclerosis,systemic sclerosis, hematological malignancies, myocardial infarction,organ transplantation rejection, chronic allograft nephropathy,cirrhosis, liver failure, heart failure, GvHD, tibial fracture, bonefactures, left ventricular dysfunction, leukemia, myelodysplasticsyndrome, Crohn's disease, diabetes, obesity, metabolic diseases anddisorders (such as lysosomal storage diseases including Tay-Sachsdisease, Gaucher disease, Pompe disease, Hurler syndrome andmetachromatic leukodystrophy), fatty liver disease, chronic obstructivepulmonary disease, osteogenesis imperfecta, homozygous familialhypocholesterolemia, hypocholesterolemia, treatment followingmeniscectomy, adult periodontitis, periodontitis, vasculogenesis inpatients with severe myocardial ischemia, spinal cord injury,osteodysplasia, critical limb ischemia, diabetic foot disease, primarySjogren's syndrome, osteoarthritis, cartilage defects, laminitis,multisystem atrophy, amyotropic lateral sclerosis, cardiac surgery,systemic lupus erythematosis, living kidney allografts, nonmalignant redblood cell disorders, thermal burn, radiation burn, Parkinson's disease,microfractures, epidermolysis bullosa, severe coronary ischemia,idiopathic dilated cardiomyopathy, osteonecrosis femoral head, lupusnephritis, bone void defects, ischemic cerebral stroke, after stroke,acute radiation syndrome, pulmonary disease, arthritis, boneregeneration, uveitis or combinations thereof. The MSCs of the invention(including formulations or preparations thereof) may be used to treatrespiratory conditions, particularly those including inflammatorycomponents or acute injury, such as Adult Respiratory Distress Syndrome,post-traumatic Adult Respiratory Distress Syndrome, transplant lungdisease, Chronic Obstructive Pulmonary Disease, emphysema, chronicobstructive bronchitis, bronchitis, an allergic reaction, damage due tobacterial or viral pneumonia, asthma, exposure to irritants, and tobaccouse. Additionally, the MSCs of the invention (including formulations orpreparations thereof) may be used to treat atopic dermatitis, allergicrhinitis, hearing loss (particularly autoimmune hearing loss ornoise-induced hearing loss), psoriasis. Additionally, the subject MSC(including formulations or preparations thereof) may be useful to treator prevent pain, heat sensitivity, and/or cold sensitivity.

The invention further encompasses kits comprising the mesenchymalstromal cells or preparation of mesenchymal stromal cells describedherein. The kits may comprise mesenchymal stromal cells or preparationsof mesenchymal stromal cells that are frozen or cryopreserved. Themesenchymal stromal cells or preparation of mesenchymal stromal cellscomprised in the kit may be contained in a cell delivery vehicle.

Moreover, the invention contemplates methods for treating a disease ordisorder, comprising administering an effective amount of mesenchymalstromal cells or a preparation of mesenchymal stromal cells describedherein to a subject in need thereof. The method may further comprise thetransplantation of other cells or tissues, e.g., retinal, RPE, corneal,neural, immune, bone marrow, liver or pancreatic cells. Exemplarydiseases or disorders treated include, but are not limited to, multiplesclerosis, systemic sclerosis, hematological malignancies, myocardialinfarction, organ transplantation rejection, chronic allograftnephropathy, cirrhosis, liver failure, heart failure, GvHD, tibialfracture, bone factures, left ventricular dysfunction, leukemia,myelodysplastic syndrome, Crohn's disease, diabetes, obesity, metabolicdiseases and disorders (such as lysosomal storage diseases includingTay-Sachs disease, Gaucher disease, Pompe disease, Hurler syndrome andmetachromatic leukodystrophy), fatty liver disease, chronic obstructivepulmonary disease, osteogenesis imperfecta, homozygous familialhypocholesterolemia, hypocholesterolemia, treatment followingmeniscectomy, adult periodontitis, periodontitis, vasculogenesis inpatients with severe myocardial ischemia, spinal cord injury,osteodysplasia, critical limb ischemia, diabetic foot disease, primarySjogren's syndrome, osteoarthritis, cartilage defects, laminitis,multisystem atrophy, amyotropic lateral sclerosis, cardiac surgery,refractory systemic lupus erythematosis, living kidney allografts,nonmalignant red blood cell disorders, thermal burn, radiation burn,Parkinson's disease, microfractures, epidermolysis bullosa, severecoronary ischemia, idiopathic dilated cardiomyopathy, osteonecrosisfemoral head, lupus nephritis, bone void defects, ischemic cerebralstroke, after stroke, acute radiation syndrome, pulmonary disease,arthritis, bone regeneration, or combinations thereof. In one aspect,the disease or disorder is uveitis. In another aspect, the disease ordisorder is an autoimmune disorder, e.g., multiple sclerosis, or animmune reaction against allogeneic cells. Additional exemplary diseasesor disorders treated include, but are not limited to, pain, heatsensitivity, and/or cold sensitivity.

In another aspect the present disclosure provides a pharmaceuticalpreparation as described herein comprising an amount of mesenchymalstromal cells effective to treat pain, heat sensitivity, or coldsensitivity in a subject in need thereof.

In another aspect the present disclosure provides a method of treating apain, heat sensitivity, or cold sensitivity, the method comprisingadministering mesenchymal stromal cells, such as mesenchymal stromalcells produced as described herein, to a subject in need thereof.

The invention further encompasses methods of treating bone loss orcartilage damage comprising administering an effective amount ofmesenchymal stromal cells or preparation of mesenchymal stromal cellsdescribed herein to a subject in need thereof. The mesenchymal stromalcells may be administered in combination with an allogeneic or syngeneictransplanted cell or tissue, e.g., retinal pigment epithelium cell,retinal cell, corneal cell, or muscle cell.

The present invention comprises methods of culturing hemangioblasts thatgenerate preparations MSCs, which retain potency, despite increasingnumbers of population doublings. The pharmaceutical preparations ofmesenchymal stromal cells of the present invention demonstrate improvedtherapeutic properties when administered to a mammalian host in need ofsuch administration.

In certain embodiments, the MSC preparation is characterized by the MSCsexpressing TNF-alpha receptor Type I in an amount of at least 13 pg/10⁶cells, and even more preferably at least 15 pg/10⁶ cells, 18 pg/10⁶cells or even 20 pg/10⁶ cells.

In certain embodiments, the MSCs of the present invention secrete IL6protein at a lower level than the same number of BM-MSCs, both in naïvecultures and activated by PHA. For instance, the concentration of IL-6protein in culture may be more than 10% less from the MSCs of thepresent invention when compared to the same number of BM-MSCs from young(20 to 30 years old) donors, and even more preferably more than 20%,30%, 40% or even 50% less. IL-6 levels can be analyzed, for example,using readily available ELISA kits (such as available from Mabtech,Nacka Strand, Sweden) following the manufacturer's instructions. IL-6can be assessed, to further illustrate, in the supernatants following 24and 48 h of co-culture with PBMC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of the FM-MA09-MSC line from pluripotent cells. Thisfigure shows a microscopic view of generating mesenchymal stromal cellsfrom the MA09 ESC via hemangioblasts.

FIG. 2. A phenotype of FM-MA09-MSC obtained from pluripotentcell-derived hemangioblasts. This figure shows the percentage of cellspositive for MSC surface markers in the initial hemangioblast population(left side of graph, day 7-11 hemangioblast) and after culturinghemangioblasts on Matrigel coated plates (right side of graph) and amicroscopic view of the mesenchymal stromal cells derived from thehemangioblasts (right panel photograph).

FIG. 3. Phenotypes of mesenchymal stromal cells derived from differentculture methods. This figure shows the percentage of cells positive forMSC surface markers after culturing human embryonic stem cells (ESC) ongelatin coated plates (left panel), ESC on Matrigel coated plates(middle panel), and hemangioblasts on Matrigel coated plates (rightpanel).

FIG. 4. Mesenchymal stromal cell yield from pluripotent cells. Thisfigure shows the yields of cells positive for MSC surface markersobtained from culturing ESC on gelatin coated plates (first column—noyield). ESC on Matrigel coated plates (second column), andhemangioblasts on Matrigel coated plates (third column).

FIG. 5. Acquisition of mesenchymal stromal cell markers. This figuredepicts the time for MSC surface markers to be acquired usinghemangioblasts (top line) and ESC (lower line).

FIG. 6. Phenotypes of mesenchymal stromal cells derived from differentculture methods. This figure shows the percentage of cells positive forMSC markers and negative for hematopoiesis and endothelial markers afterculturing ESC on Matrigel coated plates (left panel) and hemangioblastson Matrigel coated plates (right panel).

FIG. 7. FM-MA09-MSC display differentiation capabilities. This figuredepicts the differentiation capabilities of mesenchymal stromal cellsderived from hemangioblasts differentiated from MA09 ESC to formadipocytes and osteocytes.

FIG. 8. MSC chondrogenic differentiation. This figure depictschondrogenic differentiation of MA09 ESC hemangioblast-derivedmesenchymal stromal cells by mRNA expression of Aggrecan (chondroitinproteoglycan sulfate 1) and Collagen IIa.

FIG. 9. Transient expression of CD309 by FM-MA09-MSC. This figure showsthe transient expression of the cell surface marker CD309.

FIG. 10A. T cell proliferation in response to mitogen is suppressed byFM-MA09-MSC. This figure shows hemangioblast-derived mesenchymal stromalcells suppression of T cell proliferation caused by chemical stimulation(PMA/ionomycin).

FIG. 10B. T cell proliferation in response to antigen presenting cellsis suppressed by FM-MAW-MSC. This figure shows hemangioblast-derivedmesenchymal stromal cells suppression of T cell proliferation caused byexposure to dendritic cells.

FIGS. 11A and 11B. T cell proliferation in response to antigenpresenting cells is suppressed by FM-MA09-MSC. FIG. 11A shows thathemangioblast-derived mesenchymal stromal cells were able to increasethe percentage of CD4/CD25 double positive Tregs that are induced inresponse to IL2 stimulus.

FIG. 11B shows that hemangioblast-derived mesenchymal stromal cellsinhibit Th1 secretion of IFNγ.

FIG. 12. Proinflammatory cytokine IFNg stimulates changes in FM-MA09-MSCsurface marker expression. This figure shows that interferon gammastimulates changes in MSC surface marker expression and may enhance MSCimmunosuppressive effects.

FIG. 13. Increased potency, greater inhibitory effects of FM-MA09-MSCsas compared to BM-MSCs. FM-MA09-MSCs exert greater inhibitory effects onT cell proliferation than do BM-MSCs. (A.) Increasing the amount of MSCsin co-culture with PBMCs causes a dose-dependent reduction in T cellproliferation in response to PMA and ionomycin. Young (p4) FM-MA09-MSCsare the most potent of all cell types tested. (B.) FM-MA09-MSCs inhibitT cell proliferation to a greater degree than do BM-MSCs in response toPHA. A 5:1 ratio of PBMCs:MSCs were co-cultured for 6 days. (C.)FM-MA09-MSCs inhibit T cell proliferation in response to increasingamounts of dendritic cells better than do BM-MSCs. In (A-C), percent Tcell proliferation was assessed by BrdU incorporation in the CD4+ and/orCD8+ cell population.

FIG. 14. FM-MA09-MSCs enhance Treg induction: early passage MSCs havegreater effects than do late passage MSCs. Non-adherent PBMCs (differentdonors) were cultured+/−IL2 for 4 days in the absence or presence ofFM-MA09-MSCs. The percentage of CD4/CD25 double positive Tregs wasassessed by flow cytometry. Young (p6) or old (p16-18) FM-MA09-MSCs wereused. The black bars indicate the average of 6 experiments. MSCs as awhole had a statistically significant effect on induction of Tregs.(p=0.02).

FIG. 15. Enhanced Treg expansion by FM-MA09-MSCs as compared to BM-MSCs.FM-MA09-MSCs induce Treg expansion better than do BM-MSCs. (A.) Foldincrease in CD4/CD25 double positive Tregs. The minus IL2 condition wasset to 1 and other groups are expressed as fold induction over thislevel. MM=MA09-MSCs, BM=bone marrow MSCs. “p”=passage number. (B.) FMMA09-MSCs (MM) induce CD4/CD25/FoxP3 triple positive Tregs better thando BM-MSCs. (C.) Percent of responding PBMCs that are CD4+ areconsistent among the different treatment groups. (D.) Percent ofresponding PBMCs that are CD25+ vary among the different treatmentgroups. FM-MA09-MSCs induce greater expression of CD25 than do BM-MSCs.This difference may explain the difference in induction of Tregs.

FIG. 16. FM-MA09-MSCs have greater proliferative capacity than BM-MSCs.FM-MA09-MSCs have a greater proliferative capacity than do BM-MSCs.Cumulative population doublings are plotted against the number of daysin culture. After initial plating of ESC-derived hemangioblasts or bonemarrow-derived mononuclear cells, adherent cells were considered p0MSCs. Successive MSC passages were replated at a density of 7000cells/sq cm and harvested when the cultures were approximately 70%confluent (every 3-5 days).

FIG. 17. Process of FM-MA09-MSC generation; Matrigel effect. Removingcells from Matrigel at an early passage (i.e., p2) may temporarily slowMSC growth as compared to those maintained on Matrigel until p6.

FIG. 18. BM-MSCs and FM-MA09-MSCs undergo chondrogenesis. Safranin 0staining (indicative of cartilaginous matrix deposition) was performedon paraffin-embedded pellet mass cultures after 21 days. Images are 40×magnification.

FIG. 19. In the basal state, FM-MA09-MSCs secrete less PGE2 than doBM-MSCs yet the fold increase upon IFNγ or TNFα stimulation is greater.(A.) The amount of prostaglandin E2 secretion (pg/ml) is shown forBM-MSCs versus FM-MA09-MSCs under basal or various stimulationconditions. PGE2 amounts are normalized to cell number. (B.) Basal PGE2values are set to 1 (black line) and PGE2 secretion under variousstimuli are expressed as fold increase over basal level.

FIG. 20. FM-MA09-MSCs maintain phenotype over time. Flow cytometryanalysis of different MSC populations. (A.) Cell surface markerexpression of FM-MA09-MSCs is maintained on three different substratesand compared to BM-MSCs. (B.) Cell surface marker expression ofFM-MA09-MSCs is evaluated over time (with successive passages, asindicated).

FIG. 21. FM-MA09-MSCs express less Stro-1 and more CD10 as compared toBM-MSCs. Flow cytometry analysis of different MSC populations. Stro-1expression is lower in FM-MA09-MSCs than in BM-MSCs at the indicatedpassage number. CD10 expression is higher in FM-MA09-MSCs than inBM-MSCs. Other markers are the same for both MSC populations.

FIG. 22. Stro-1 and CD10 expression in 10 different lots of earlypassage FM-MA09-MSCs consistently show low Stro-1 and mid-range CD10expression. Flow cytometry analysis of different MSC populations. Tendifferent lots of FM-MA09-MSCs were evaluated at the indicated passagenumber for expression of Stro-1 and CD10. Stro-1 expression isconsistently low in the different lots of FM-MA09-MSCs (average of5-10%). CD10 expression is consistently at amid-range level in thedifferent lots of FM-MA09-MSCs (average of approximately 40%).

FIG. 23. FM-MA09-MSCs maintain their size as they age in culture whileBM-MSC cell size increases with age. Forward scatter/side scatter dotplots on flow cytometry (shown on the left) were used to capture thesize of MSCs. The percentage of cells in the upper right quadrant“large” cells were monitored and are displayed in the bar graph.

FIG. 24. CD10 and CD24 are upregulated in FM-MA09-MSCs as compared toBM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCsin the basal state. Quantitative RT-PCR with Taqman probes was used toassess the expression of the indicated genes and normalized to twohousekeeping genes. The average of quadruplicate readings is shown+/−standard deviation.

FIG. 25. Aire-1 and IL-11 are upregulated in FM-MA09-MSCs as compared toBM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCsin the basal state. Quantitative RT-PCR with Taqman probes was used toassess the expression of the indicated genes and normalized to twohousekeeping genes. The average of quadruplicate readings is shown+/−standard deviation.

FIG. 26. Ang-1 and CXCL1 are upregulated in FM-MA09-MSCs as compared toBM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCsin the basal state. Quantitative RT-PCR with Taqman probes was used toassess the expression of the indicated genes and normalized to twohousekeeping genes. The average of quadruplicate readings is shown+/−standard deviation.

FIG. 27. IL6 and VEGF are downregulated in FM-MA09-MSCs as compared toBM-MSCs. Gene expression analysis is shown for BM-MSCs and FM-MA09-MSCsin the basal state. Quantitative RT-PCR with Taqman probes was used toassess the expression of the indicated genes and normalized to twohousekeeping genes. The average of quadruplicate readings is shown+/−standard deviation.

FIG. 28. FM-MA09-MSG's and BM-MSCs show increased indoleamine 2,3deoxygenase (IDO) activity in response to 3 days of IFNγ stimulation.Comparison of MSCs stimulated with 50 ng/ml IFNg for 3 days, for theirability to convert tryptophan into kynurenine (indicative of IDOactivity). For each MSC population, 1 million cells were lysed and usedin the assay.

FIG. 29. Age-related changes in FM-MA09-MSC expression of Aire-1 andPrion Protein (PrP): two proteins involved in immune suppression andproliferation, respectively. Western blot analysis of Aire-1 and PrPexpression in FM-MA09-MSCs whole cell lysates at different passagenumbers (p). Actin expression is shown as loading control. Differencesin Aire-1 and PrP expression are noted by referencing the actin loadingcontrols.

FIG. 30. FM-MA09-MSCs secrete less IL6 than BM-MSCs do in the basalstate. Cytokine arrays showing positive controls for normalization (4dots on left) and IL6 (boxed) in MSC conditioned medium. BM-MSCs fromtwo different donors are compared to 4 different lots of FM-MA09-MSCs.

FIG. 31. FM-MA09-MSCs secrete less IL6 than BM-MSCs in the basal andIFNγ-stimulated state. Cytokine arrays showing positive controls fornormalization (4 dots on left) and IL6 (boxed) in MSC conditionedmedium. Passage 7 BM-MSCs are compared to p7 FM-MA09-MSCs after 48hours+/−IFNγ treatment.

FIG. 32. FM-MA09-MSCs secrete less VEGF than BM-MSCs in the basal andIFNγ-stimulated state. Cytokine arrays showing positive controls fornormalization (4 dots on left) and VEGF (boxed) in MSC conditionedmedium. Passage 7 BM-MSCs are compared to p7 FM-MA09-MSCs after 48hours+/−IFNγ treatment.

FIG. 33A-E. MSCs are efficiently derived from hESCs. (A) Phase contrastimages for hESC (CT2) differentiation into MSCs through multiple stages.hESCs cultured on Matrige in TeSR1 medium were used to form EBs for 4days. The EB cells were then dissociated and cultured in hemangioblast(HB) Growth Medium for 6-12 days. HB-enriched cells were then harvested,rinsed, and cultured in MSC Medium for 7-14 days to form hES-MSCs. (B)Flow cytometric analyses of cell surface markers on day-9 FIB-enrichedcells and day-14 hES-MSCs (CT2). (C) Immunofluorescence for HLA-G (red)and DAPI (blue) of hES-MSCs (MA09). (D) hES-MSCs (MA09) were treated+/−IFNγ at 50 ng/ml for 7 days, and stained for HLA-ABC, HLA-DR andCD80. Bars indicate the mean of 4 independent experiments +/−SD. (E) Invitro multi-lineage differentiation of hES-MSCs (MA09) (bottom panel)with undifferentiated hES-MSCs as controls (top panel). Phase contrastimages of Alizarin Red staining for osteogenic differentiation (left),Oil Red 0 staining for adipogenic differentiation (middle), andhistological sections of chondrogenic pellets stained with Alcian Blue(right). Scale bars=50 μm.

FIG. 34A-D. hES-MSCs attenuate the disease score of EAE mice andanalyses of the CNS. (A) Disease scores of EAE mice treated with threedifferent hES-MSC lines (CT2, MA09, and H9) pre-onset. 10⁶ hES-MSCs orundifferentiated hESCs as a control were i.p injected 6 dayspost-immunization. N=5, ***P<0.001. (B) Analysis of demyelination andmicroglial responses in the CNS of EAE mice treated with hES-MSCs or PBSas a control. Immunohistochemical detection of MBP (red), CD3 for Tcells (green) and IBA1 for microglia (green) on lumbar spinal cord crosssections from EAE mice treated with either hES-MSCs (a and c) or PBS (band d). (C) Quantitative analysis of myelin in the spinal cord wasperformed using relative fluorescent intensity (RFI) measurement of MBPexpression in digitally captured spinal cord hemi-sections. N=4-6,**P<0.02. (D) Disease scores of EAE mice treated with hES-MSCspost-onset. 10⁶ hES-MSCs were i.p injected 18 days post-immunization.n=6, ***P<0.001.

FIG. 35A-B. Irradiated hES-MSCs retain anti-EAE effect. (A) IrradiatedhES-MSCs (Irr-hES-MSCs), derived from MA09 hESCs, reduced disease scorein EAE mice. PBS, 1×10⁶ hES-MSCs or 2×10⁶ Irr-hES-MSCs were i.p.injected into EAE mice 6 days post-immunization followed by diseasescoring. N=5, ***P<0.001. (B) Non-irradiated (left) and irradiated(right) luciferase-expressing hES-MSCs (CT2) were tracked in EAE mice.Luciferase signals were monitored at various days post-cell injection byinjecting the mice with D-Luciferin. Images were taken using the Xenogen1 VIS 100 system.

FIG. 36A-E. hES-MSCs have stronger anti-EAE effect in vivo than BM-MSCs.(A-C) Disease scores of EAE mice treated with PBS, BM-MSCs or hES-MSCs(MA09) pre-onset. 1×10⁶ hES-MSCs or BM-MSCs (each BM-MSC # represents adifferent donor) were i.p injected 6 days post-immunization. N=5 pergroup, ***P<0.001 between hES-MSCs and any of the three BM-MSCs treatedgroups. (D) Total numbers of CD4⁺, CD8⁺, Th1, and Th17 cells in the CNSof EAE mice treated with PBS, hES-MSCs, or BM-MSCs on day 32post-immunization. Lymphocytes purified from the CNS were analyzed fornumbers of CD4⁺ and CD8⁺ cells using flow cytometry or stimulated withTPA and ionomycin followed by intracellular staining for numbers ofIL-17⁺ and IFNγ⁺ cells (right panels). N=4, *P<0.05, **P<0.01. (E)Qualitative analysis of myelin content in spinal cord cross-sections ofEAE mice treated with PBS, BM-MSCs, or hES-MSCs using Fluoromyelin stain(green) and counterstained with DAPI (blue) to indicate infiltration ofnucleated cells.

FIG. 37A shows the rate of survival of BWF1 mice after treatment withMSCs (one dose of 2×10⁶ cells, or two doses of 0.5×10⁶ cells per dose)versus the rate of survival of control animals. Uppermost (green) line,two doses of 0.5×10⁶ MSC administered, 100% survival at 50 days posttreatment; middle (blue) line, one does of 2×10⁶ MSC administered, about95% survival at 50 days post treatment; lower (red) line, controls,about 60% survival at 50 days post treatment.

FIG. 37B shows the relative proteinuria (presence of an excess of serumproteins in the urine) for BWF1 mice after treatment with MSCs (one doseof 2×10⁶ cells, or two doses of 0.5×10⁶ cells per dose) versus controlanimals.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to methods of generating mesenchymalstromal cells, preparations of mesenchymal stromal cells from culturinghemangioblasts, methods of culturing hemangioblasts, and methods oftreating a pathology using mesenchymal stromal cells.

The methods of the instant invention, whereby hemangioblast culturesproduce increased yields of mesenchymal stromal cells, compared to priorprocesses, are more efficient than previous processes at producingsubstantially ESC-free mesenchymal stromal cells. Thehemangioblast-derived mesenchymal stromal cells of the instant inventionretain a novel, youthful phenotype as defined by expression or lackthereof of specific markers.

In certain embodiments, the MSC preparation (such as cultures having atleast 10³, 10⁴, 10⁵ or even 10⁶ MSCs) may have, as an average, telomerelengths that are at least 30 percent of the telomere length of an ESCand/or human iPS cell (or the average of a population of ESC and/orhuman iPS cells), and preferably at least 40, 50, 60, 70 80 or even 90percent of the telomere length of an ESC and/or human iPS cell (or ofthe average of a population of ESC and/or human iPS cells). For example,said ESC and/or human iPS cell (or said population of ESC and/or humaniPS cells) may be a cell or cell population from which said MSC cellswere differentiated.

The MSC preparation may, as a population, have a mean terminalrestriction fragment length (TRF) that is longer than 4 kb, andpreferably longer than 5, 6, 7, 8, 9, 10, 11, 12 or even 13 kb. In anexemplary embodiment, the MSCs of the preparation may have an averageTRF that is 10 kb or longer.

In certain embodiments, the MSC preparation (such as cultures having atleast 10³, 10⁴, 10⁵, 10⁶, 10⁷ or even 10⁸ MSCs) has a replicativelifespan that is greater than the replicative lifespan of MSCpreparations obtained from other sources (e.g., cultures derived fromdonated human tissue, such as fetal, infant, child, adolescent or adulttissue). Replicative lifespan may be assessed by determining the numberof population doublings or passages in culture prior to replicativesenescence, i.e., where more than 10, 20, 30, 40 or even 50 percent ofthe cells in culture senesce before the next doubling or passage. Forexample, the subject MSC preparations may have a replicative lifespanthat is at least 10 doublings greater than that of an MSC preparationderived from donated human tissue (particularly derived from adult bonemarrow or adult adipose tissue), and preferably at least 20, 30, 40, 50,60, 70 80, 90 or even 100 population doublings. In certain embodiments,the MSC preparations may have a replicative lifespan that permits atleast 8 passages before more than 50 percent of the cells senesce and/ordifferentiate into non-MSC cell types (such as fibroblasts), and morepreferably at least 10, 12, 14, 16, 18 or even 20 passages beforereaching that point. In certain embodiments, the MSC preparation mayhave a replicative lifespan that permits at least 2 times as manydoublings or passages relative to adult bone marrow-derived MSCpreparations and/or adipose-derived MSC preparations (e.g., equivalentstarting number of cells) before more than 50 percent of the cellssenesce and/or differentiate into non-MSC cell types (such asfibroblasts), and more preferably at least 4, 6, 8 or even 10 times asmany doublings or passages.

In certain embodiments, the MSC preparation of the present invention(such as cultures having at least 10³, 10⁴, 10⁵, 10⁶, 10⁷ or even 10⁸MSCs) have a statistically significant decreased content and/orenzymatic activity of proteins involved in cell cycle regulation andaging relative to passage 1 (P1), passage 2 (P2), passage 3 (P3),passage 4 (P4) and/or passage 5 (P5) MSC preparations derived from othersources (e.g., cultures derived from donated human tissue, such asfetal, infant, child, adolescent or adult tissue), and particularly bonemarrow-derived MSCs and adipose-derived MSCs. For example, the subjectMSC preparation has a proteasome 26S subunit, non-ATPase regulatorysubunit 11 (PSMD11) protein content that is less than 75 percent of thecontent in MSCs from donated human tissue (particularly derived fromadult bone marrow or adult adipose tissue), and even more preferablyless than 60, 50, 40, 30, 20 or even 10 percent.

In certain embodiments, the MSC preparation of the present invention(such as cultures having at least 10³, 10⁴, 10⁵, 10⁶, 10⁷ or even 10⁸MSCs) have a statistically significant decreased content and/orenzymatic activity of proteins involved in energy and/or lipidmetabolism of the cell relative to passage 1 (P1), passage 2 (P2),passage 3 (P3), passage 4 (P4) and/or passage 5 (P5) MSC preparationsderived from other sources (e.g., cultures derived from donated humantissue, such as fetal, infant, child, adolescent or adult tissue), andparticularly bone marrow-derived MSCs and adipose-derived MSCs. Toillustrate, the subject MSC preparation has a protein content that isless than 90 percent of the content in MSCs from donated human tissue(particularly derived from adult bone marrow or adult adipose tissue),and even more preferably less than 60, 50, 40, 30, 20 or even 10percent, for one or more proteins involved in metabolic pathways for ATPor NADPH synthesis such as glycolysis (such as fructose-biphosphatealdolase A, ALDOA; aldo-keto reductase family 1, member A1, AKR1A1);glyceraldehyde-3-phosphate, GAPDH), the tricarboxylic acid cycle (TCAcycle) (such as isocitrate dehydrogenase 1, IDH1), the pentose phosphatepathway (such as glucose-6-phosphate dehydrogenase, G6PD) and thebiosynthesis of UDP-glucose in the glucuronic acid biosynthetic pathway(such as UDP-glucose 6-dehydrogenase, UGDH). To further illustrate, thesubject MSC preparation has a protein content that is less than 90percent of the content in MSCs from donated human tissue (particularlyderived from adult bone marrow or adult adipose tissue), and even morepreferably less than 60, 50, 40, 30, 20 or even 10 percent, for one ormore proteins involved in lipid metabolism, such as enoyl-CoA hydratase,short chain, 1 (ECHS1) and/or acetyl-CoA acetyltransferase (ACAT2).

In certain embodiments, the MSC preparation of the present invention(such as cultures having at least 10³, 10⁴, 10⁵, 10⁶, 10⁷ or even 10⁸MSCs) have a statistically significant decreased content and/orenzymatic activity of proteins involved in apoptosis of the cellrelative to passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4(P4) and/or passage 5 (P5) MSC preparations derived from other sources(e.g., cultures derived from donated human tissue, such as fetal,infant, child, adolescent or adult tissue), and particularly bonemarrow-derived MSCs and adipose-derived MSCs. To illustrate, the subjectMSC preparation has a protein content that is less than 90 percent ofthe content in MSCs from donated human tissue (particularly derived fromadult bone marrow or adult adipose tissue), and even more preferablyless than 60, 50, 40, 30, 20 or even 10 percent, for one or moreproteins annexin A1 (ANXA1), A2 (ANXA2), A5 (ANXA5), thevoltage-dependent anion-selective channel protein 1 (VDAC1), and/orglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Without being bound by theory, it is believed that the statisticallysignificant difference in content and/or enzymatic activity of proteinsinvolved in energy and/or lipid metabolism and/or apotosis of the celldisplayed by the hemangioblast-derived MSCs of the present invention isattributable, at least in part, to the homogeneous nature of thepreparations. For example, hemangioblast-derived MSCs of the presentinvention have homogeneous MHC gene expression, i.e., completely MHCmatched, unlike adult derived MSC banks, in which the cells are derivedfrom multiple different donors, i.e., MHC mismatched. A therapeutic doseof MSCs is about 2-8 million cells/kg (or about 130-500 million cellsper dose).

Definitions

“Pluripotent cells” and “pluripotent stem cells” as used herein, refersbroadly to a cell capable of prolonged or virtually indefiniteproliferation in vitro while retaining their undifferentiated state,exhibiting a stable (preferably normal) karyotype, and having thecapacity to differentiate into all three germ layers (i.e., ectoderm,mesoderm and endoderm) under the appropriate conditions. Typicallypluripotent cells (a) are capable of inducing teratomas whentransplanted in immunodeficient (SLID) mice; (b) are capable ofdifferentiating to cell types of all three germ layers (e.g.,ectodermal, mesodermal, and endodermal cell types); and (c) express atleast one hES cell marker (such as Oct-4, alkaline phosphatase, SSEA 3surface antigen, SSEA 4 surface antigen, NANOG, TRA 1 60, TRA 1 81,SOX2, REX1). Exemplary pluripotent cells may express Oct-4, alkalinephosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1 60,and/or TRA 1 81. Additional exemplary pluripotent cells include but arenot limited to embryonic stem cells, induced pluripotent cells (iPS)cells, embryo-derived cells, pluripotent cells produced from embryonicgerm (EG) cells (e.g., by culturing in the presence of FGF-2, LIF andSCF), parthenogenetic ES cells, ES cells produced from cultured innercell mass cells (ICM), ES cells produced from a blastomere, and ES cellsproduced by nuclear transfer (e.g., a somatic cell nucleus transferredinto a recipient oocyte). Exemplary pluripotent cells may be producedwithout destruction of an embryo. For example, induced pluripotent cellsmay be produced from cells obtained without embryo destruction. As afurther example, pluripotent cells may be produced from a biopsiedblastomere (which can be accomplished without harm to the remainingembryo); optionally, the remaining embryo may be cryopreserved,cultured, and/or implanted into a suitable host. Pluripotent cells (fromwhatever source) may be genetically modified or otherwise modified toincrease longevity, potency, homing, or to deliver a desired factor incells that are differentiated from such pluripotent cells (for example,MSCs, and hemangioblasts). As non-limiting examples thereof, thepluripotent cells may be genetically modified to express Sirt1 (therebyincreasing longevity), express one or more telomerase subunit genesoptionally under the control of an inducible or repressible promoter,incorporate a fluorescent label, incorporate iron oxide particles orother such reagent (which could be used for cell tracking via in vivoimaging, MRI, etc., see Thu et al., Nat Med. 2012 Feb. 26; 18(3):463-7),express bFGF which may improve longevity (see Go et al., J. Biochem.142, 741-748 (2007)), express CXCR4 for homing (see Shi et al.,Haematologica. 2007 July; 92(7):897-904), express recombinant TRAIL toinduce caspase-mediated apoptosis in cancer cells like Gliomas (seeSasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24; 106(12):4822-7),etc.

“Embryo” or “embryonic,” as used herein refers broadly to a developingcell mass that has not implanted into the uterine membrane of a maternalhost. An “embryonic cell” is a cell isolated from or contained in anembryo. This also includes blastomeres, obtained as early as thetwo-cell stage, and aggregated blastomeres.

“Embryonic stem cells” (ES cells or ESC) encompasses pluripotent cellsproduced from embryonic cells (such as from cultured inner cell masscells or cultured blastomeres) as well as induced pluripotent cells(further described below). Frequently such cells are or have beenserially passaged as cell lines. Embryonic stem cells may be used as apluripotent stem cell in the processes of producing hemangioblasts asdescribed herein. For example, ES cells may be produced by methods knownin the art including derivation from an embryo produced by any method(including by sexual or asexual means) such as fertilization of an eggcell with sperm or sperm DNA, nuclear transfer (including somatic cellnuclear transfer), or parthenogenesis. As a further example, embryonicstem cells also include cells produced by somatic cell nuclear transfer,even when non-embryonic cells are used in the process. For example, EScells may be derived from the ICM of blastocyst stage embryos, as wellas embryonic stem cells derived from one or more blastomeres. Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, and androgenesis. As further discussedabove (see “pluripotent cells), ES cells may be genetically modified orotherwise modified to increase longevity, potency, homing, or to delivera desired factor in cells that are differentiated from such pluripotentcells (for example, MSCs, and hemangioblasts).

ES cells may be generated with homozygosity or hemizygosity in one ormore HLA genes, e.g., through genetic manipulation, screening forspontaneous loss of heterozygosity, etc. ES cells may be geneticallymodified or otherwise modified to increase longevity, potency, homing,or to deliver a desired factor in cells that are differentiated fromsuch pluripotent cells (for example, MSCs and hemangioblasts). Embryonicstem cells, regardless of their source or the particular method used toproduce them, typically possess one or more of the following attributes:(i) the ability to differentiate into cells of all three germ layers,(ii) expression of at least Oct-4 and alkaline phosphatase, and (iii)the ability to produce teratomas when transplanted intoimmunocompromised animals. Embryonic stem cells that may be used inembodiments of the present invention include, but are not limited to,human ES cells (“hESC” or “hES cells”) such as CT2, MA01, MA09, ACT-4,No. 3, H1, H7, H9, H14 and ACT30 embryonic stem cells. Additionalexemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. Seealso NIH Human Embryonic Stem Cell Registry. An exemplary humanembryonic stem cell line that may be used is MA09 cells. The isolationand preparation of MA09 cells was previously described in Klimanskaya,et al. (2006) “Human Embryonic Stem Cell lines Derived from SingleBlastomeres.” Nature 444: 481-485. The human ES cells used in accordancewith exemplary embodiments of the present invention may be derived andmaintained in accordance with GMP standards.

Exemplary hES cell markers include but are not limited to: alkalinephosphatase, Oct-4, Nanog, Stage-specific embryonic antigen-3 (SSEA-3),Stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81,TRA-2-49/6E, Sox2, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4,E-CADHERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2,Genesis, Germ cell nuclear factor, and Stem cell factor (SCF or c-Kitligand). Additionally, embryonic stem cells may express Oct-4, alkalinephosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1 60,and/or TRA 1 81.

The ESCs may be initially co-cultivated with murine embryonic feedercells (MEF) cells. The MEF cells may be mitotically inactivated byexposure to mitomycin C prior to seeding ESCs in co culture, and thusthe MEFs do not propagate in culture. Additionally, ESC cell culturesmay be examined microscopically and colonies containing non ESC cellmorphology may be picked and discarded, e.g., using a stem cell cuttingtool, by laser ablation, or other means. Typically, after the point ofharvest of the ESCs for seeding for embryoid body formation noadditional MEF cells are used.

“Embryo-derived cells” (EDC), as used herein, refers broadly topluripotent morula-derived cells, blastocyst-derived cells includingthose of the inner cell mass, embryonic shield, or epiblast, or otherpluripotent stem cells of the early embryo, including primitiveendoderm, ectoderm, and mesoderm and their derivatives. “EDC” alsoincluding blastomeres and cell masses from aggregated single blastomeresor embryos from varying stages of development, but excludes humanembryonic stem cells that have been passaged as cell lines.

Exemplary ESC cell markers include but are not limited to: alkalinephosphatase, Oct-4, Nanog, Stage-specific embryonic antigen-3 (SSEA-3),Stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81,TRA-2-4916E, Sox2, growth and differentiation factor 3 (GDF3), reducedexpression 1 (REX1), fibroblast growth factor 4 (FGF4), embryoniccell-specific gene 1 (ESG1), developmental pluripotency-associated 2(DPPA2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4,E-CADHERIN, Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2,Genesis, Germ cell nuclear factor, and Stem cell factor (SCF or c-Kitligand).

“Potency”, as used herein, refers broadly to the concentration, e.g.,molar, of a reagent (such as hemangioblast-derived MSCs) that produces adefined effect. Potency may be defined in terms of effectiveconcentration (EC50), which does not involve measurements of maximaleffect but, instead, the effect at various locations along theconcentration axis of dose response curves. Potency may also bedetermined from either graded (EC50) or quantal dose-response curves(ED50, TD50 and LD50); however, potency is preferably measured by EC50.The term “EC50” refers to the concentration of a drug, antibody ortoxicant which induces a response halfway between the baseline andmaximum effect after some specified exposure time. The EC50 of a gradeddose response curve therefore represents the concentration of a compoundwhere 50% of its maximal effect is observed. The EC50 of a quantal doseresponse curve represents the concentration of a compound where 50% ofthe population exhibit a response, after a specified exposure duration.The EC50 may be determined using animal studies in which a definedanimal model demonstrates a measurable, physiological change in responseto application of the drug; cell-based assays that use a specified cellsystem, which on addition of the drug, demonstrate a measureablebiological response; and/or enzymatic reactions where the biologicalactivity of the drug can be measured by the accumulation of productfollowing the chemical reaction facilitated by the drug. Preferably, animmune regulatory assay is used to determine EC50. Non-limiting examplesof such immune regulatory assays include intracellular cytokine,cytotoxicity, regulatory capacity, cell signaling capacity,proliferative capacity, apoptotic evaluations, and other assays.

“Mesenchymal stem cells” (MSC) as used herein refers to multipotent stemcells with self-renewal capacity and the ability to differentiate intoosteoblasts, chondrocytes, and adipocytes, among other mesenchymal celllineages. In addition to these characteristics, MSCs may be identifiedby the expression of one or more markers as further described herein.Such cells may be used to treat a range of clinical conditions,including immunological disorders as well as degenerative diseases suchas graft-versus-host disease (GVHD), myocardial infarction andinflammatory and autoimmune diseases and disorders, among others. Exceptwhere the context indicates otherwise, MSCs may include cells from adultsources and cord blood. MSCs (or a cell from which they are generated,such as a pluripotent cell) may be genetically modified or otherwisemodified to increase longevity, potency, homing, or to deliver a desiredfactor in the MSCs or cells that are differentiated from such MSCs. Asnon-limiting examples thereof, the MSCs cells may be geneticallymodified to express Sirt1 (thereby increasing longevity), express one ormore telomerase subunit genes optionally under the control of aninducible or repressible promoter, incorporate a fluorescent label,incorporate iron oxide particles or other such reagent (which could beused for cell tracking via in vivo imaging, MRI, etc., see Thu et al.,Nat Med. 2012 Feb. 26; 18(3):463-7), express bFGF which may improvelongevity (see Go et al., J. Biochem. 142, 741-748 (2007)), expressCXCR4 for homing (see Shi et al., Haematologica. 2007 July;92(7):897-904), express recombinant TRAIL to induce caspase-mediatedxapoptosis in cancer cells like Gliomas (see Sasportas et al., Proc NatlAcad Sci USA. 2009 Mar. 24; 106(12):4822-7), etc.

“Therapy,” “therapeutic,” “treating,” “treat” or “treatment”, as usedherein, refers broadly to treating a disease, arresting or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.“Therapy”, “therapeutic,” “treating,” “treat” or “treatment” encompassesprophylaxis, prevention, treatment, cure, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. “Therapy”, “therapeutic,” “treating,” “treat” or“treatment” encompasses an alleviation of signs and/or symptoms inpatients with ongoing disease signs and/or symptoms (e.g., muscleweakness, multiple sclerosis.) “Therapy”, “therapeutic,” “treating,”“treat” or “treatment” also encompasses “prophylaxis” and “prevention”.Prophylaxis includes preventing disease occurring subsequent totreatment of a disease in a patient or reducing the incidence orseverity of the disease in a patient. The term “reduced”, for purpose oftherapy, “therapeutic,” “treating,” “treat” or “treatment” refersbroadly to the clinical significant reduction in signs and/or symptoms.“Therapy”, “therapeutic,” “treating,” “treat” or “treatment” includestreating relapses or recurrent signs and/or symptoms (e.g., retinaldegeneration, loss of vision.) “Therapy”, “therapeutic,” “treating,”“treat” or “treatment” encompasses but is not limited to precluding theappearance of signs and/or symptoms anytime as well as reducing existingsigns and/or symptoms and eliminating existing signs and/or symptoms.“Therapy”, “therapeutic,” “treating,” “treat” or “treatment” includestreating chronic disease (“maintenance”) and acute disease. For example,treatment includes treating or preventing relapses or the recurrence ofsigns and/or symptoms (e.g., muscle weakness, multiple sclerosis).

“Normalizing a pathology”, as used herein, refers to reverting theabnormal structure and/or function resulting from a disease to a morenormal state. Normalization suggests that by correcting theabnormalities in structure and/or function of a tissue, organ, celltype, etc. resulting from a disease, the progression of the pathologycan be controlled and improved. For example, following treatment withthe ESC-MSCs of the present invention the abnormalities of the immunesystem as a result of autoimmune disorders, e.g., MS, may be improved,corrected, and/or reversed.

Induced Pluripotent Stem Cells

Further exemplary pluripotent stem cells include induced pluripotentstem cells (iPS cells) generated by reprogramming a somatic cell byexpressing or inducing expression of a combination of factors(“reprogramming factors”). iPS cells may be generated using fetal,postnatal, newborn, juvenile, or adult somatic cells. iPS cells may beobtained from a cell bank. Alternatively, iPS cells may be newlygenerated (by processes known in the art) prior to commencingdifferentiation to RPE cells or another cell type. The making of iPScells may be an initial step in the production of differentiated cells.iPS cells may be specifically generated using material from a particularpatient or matched donor with the goal of generating tissue-matched RPEcells. iPS cells can be produced from cells that are not substantiallyimmunogenic in an intended recipient, e.g., produced from autologouscells or from cells histocompatible to an intended recipient. As furtherdiscussed above (see “pluripotent cells”), pluripotent cells includingiPS cells may be genetically modified or otherwise modified to increaselongevity, potency, homing, or to deliver a desired factor in cells thatare differentiated from such pluripotent cells (for example, MSCs andhemangioblasts).

As a further example, induced pluripotent stem cells may be generated byreprogramming a somatic or other cell by contacting the cell with one ormore reprogramming factors. For example, the reprogramming factor(s) maybe expressed by the cell, e.g., from an exogenous nucleic acid added tothe cell, or from an endogenous gene in response to a factor such as asmall molecule, microRNA, or the like that promotes or inducesexpression of that gene (see Suh and Blelloch, Development 138,1653-1661 (2011); Miyosh et al., Cell Stem Cell (2011),doi:10.1016/j.stem.2011.05.001; Sancho-Martinez et al., Journal ofMolecular Cell Biology (2011) 1-3; Anokye-Danso et al., Cell Stem Cell8, 376-388, Apr. 8, 2011; Orkin and Hochedlinger, Cell 145, 835-850,Jun. 10, 2011, each of which is incorporated by reference herein in itsentirety). Reprogramming factors may be provided from an exogenoussource, e.g., by being added to the culture media, and may be introducedinto cells by methods known in the art such as through coupling to cellentry peptides, protein or nucleic acid transfection agents,lipofection, electroporation, biolistic particle delivery system (genegun), microinjection, and the like. iPS cells can be generated usingfetal, postnatal, newborn, juvenile, or adult somatic cells. In certainembodiments, factors that can be used to reprogram somatic cells topluripotent stem cells include, for example, a combination of Oct4(sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4. In otherembodiments, factors that can be used to reprogram somatic cells topluripotent stem cells include, for example, a combination of Oct-4,Sox2, Nanog, and Lin28. In other embodiments, somatic cells arereprogrammed by expressing at least 2 reprogramming factors, at leastthree reprogramming factors, or four reprogramming factors. In otherembodiments, additional reprogramming factors are identified and usedalone or in combination with one or more known reprogramming factors toreprogram a somatic cell to a pluripotent stem cell. iPS cells typicallycan be identified by expression of the same markers as embryonic stemcells, though a particular iPS cell line may vary in its expressionprofile.

The induced pluripotent stem cell may be produced by expressing orinducing the expression of one or more reprogramming factors in asomatic cell. The somatic cell is a fibroblast, such as a dermalfibroblast, synovial fibroblast, or lung fibroblast, or anon-fibroblastic somatic cell. The somatic cell is reprogrammed byexpressing at least 1, 2, 3, 4, 5 reprogramming factors. Thereprogramming factors may be selected from Oct 3/4, Sox2, NANOG, Lin28,c Myc, and Klf4. Expression of the reprogramming factors may be inducedby contacting the somatic cells with at least one agent, such as a smallorganic molecule agents, that induce expression of reprogrammingfactors.

The somatic cell may also be reprogrammed using a combinatorial approachwherein the reprogramming factor is expressed (e.g., using a viralvector, plasmid, and the like) and the expression of the reprogrammingfactor is induced (e.g., using a small organic molecule.) For example,reprogramming factors may be expressed in the somatic cell by infectionusing a viral vector, such as a retroviral vector or a lentiviralvector. Also, reprogramming factors may be expressed in the somatic cellusing a non-integrative vector, such as an episomal plasmid. See, e.g.,Yu et al., Science. 2009 May 8; 324(5928):797-801, which is herebyincorporated by reference in its entirety. When reprogramming factorsare expressed using non-integrative vectors, the factors may beexpressed in the cells using electroporation, transfection, ortransformation of the somatic cells with the vectors. For example, inmouse cells, expression of four factors (Oct3/4, Sox2, c myc, and Klf4)using integrative viral vectors can be used to reprogram a somatic cell.In human cells, expression of four factors (Oct34, Sox2, NANOG, andLin28) using integrative viral vectors can be used to reprogram asomatic cell.

Once the reprogramming factors are expressed in the cells, the cells maybe cultured. Over time, cells with ES characteristics appear in theculture dish. The cells may be chosen and subcultured based on, forexample, ES morphology, or based on expression of a selectable ordetectable marker. The cells may be cultured to produce a culture ofcells that resemble ES cells—these are putative iPS cells. iPS cellstypically can be identified by expression of the same markers as otherembryonic stem cells, though a particular iPS cell line may vary in itsexpression profile. Exemplary iPS cells may express Oct-4, alkalinephosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1 60,anchor TRA 1 81.

To confirm the pluripotency of the iPS cells, the cells may be tested inone or more assays of pluripotency. For example, the cells may be testedfor expression of ES cell markers; the cells may be evaluated forability to produce teratomas when transplanted into SCUD mice; the cellsmay be evaluated for ability to differentiate to produce cell types ofall three germ layers. Once a pluripotent iPS cell is obtained it may beused to produce hemangioblast and MSC cells.

Hemangioblasts

Hemangioblasts are multipotent and serve as the common precursor to bothhematopoietic and endothelial cell lineages. During embryonicdevelopment, they are believed to arise as a transitional cell type thatemerges during early mesoderm development and colonizes primitive bloodislands (Choi et al. Development 125 (4): 725-732 (1998). Once there,hemangioblasts are capable of giving rise to both primitive anddefinitive hematopoietic cells, HSCs, and endothelial cells (Mikkola etal, J. Hematother. Stem Cell Res 11(1): 9-17 (2002).

Hemangioblasts may be derived in vitro from both mouse ESCs (Kennedy etal, Nature (386): 488-493 (1997); Perlingeiro et al, Stem Cells (21):272-280 (2003)) and human ESCs (ref. 14, 15, Yu et al., Blood 2010 116:4786-4794). Other studies claim to have isolated hemangioblasts fromumbilical cord blood (Bordoni et al, Hepatology 45 (5) 1218-1228),circulating CD34− lin− CD45− CD133− cells from peripheral blood (Ciraciet al, Blood 118: 2105-2115), and from mouse uterus (Sun et al, Blood116 (16): 2932-2941 (2010)). Both mouse and human ESC-derivedhemangioblasts have been obtained through the culture anddifferentiation of clusters of cells grown in liquid culture followed bygrowth of the cells in semi-solid medium containing various cytokinesand growth factors (Kennedy, Perlingeiro, ref 14, 15); see also, U.S.Pat. No. 8,017,393, which is hereby incorporated by reference in itsentirety. For the purposes of this application, the term hemangioblastsalso includes the hemangio-colony forming cells described in U.S. Pat.No. 8,017,393, which in addition to being capable of differentiatinginto hermatopoietic and endothelial cell lineages, are capable ofbecoming smooth muscle cells and which are not positive for CD34, CD31,KDR, and CD133. Hemangioblasts useful in the methods described hereinmay be derived or obtained from any of these known methods. For example,embryoid bodies may be formed by culturing pluripotent cells undernon-attached conditions, e.g., on a low-adherent substrate or in a“hanging drop.” In these cultures, ES cells can form clumps or clustersof cells denominated as embryoid bodies. See Itskovitz-Eldor et al., MolMed. 2000 February; 6(2):88-95, which is hereby incorporated byreference in its entirety. Typically, embryoid bodies initially form assolid clumps or clusters of pluripotent cells, and over time some of theembryoid bodies come to include fluid filled cavities, the latter formerbeing referred to in the literature as “simple” EBs and the latter as“cystic” embryoid bodies. Id. The cells in these EBs (both solid andcystic forms) can differentiate and over time produce increasing numbersof cells. Optionally EBs may then be cultured as adherent cultures andallowed to form outgrowths. Likewise, pluripotent cells that are allowedto overgrow and form a multilayer cell population can differentiate overtime.

In one embodiment, hemangioblasts are generated by the steps comprising(a) culturing an ESC line for 2, 3, 4, 5, 6 or 7 days to form clustersof cells, and (b) inducing said clusters of cells to differentiate intohemangioblasts. In a further embodiment, the clusters of cells in step(b) of are cultured in a cytokine-rich serum-free methylcellulose basedmedium (14, 15).

In one embodiment, hemangioblasts are generated by the steps comprising(a) culturing an ESC line selected from the group consisting of CT2,MA09, H7, H9, MA01, HuES3, H1gfp, NED1, NED2, NED3, NED4, NED5, and NED7for 2, 3, 4, 5, 6 or 7 days to form clusters of cells, and (b) inducingsaid clusters of cells to differentiate into hemangioblasts by culturingin a cytokine-rich, serum-free, methylcellulose based medium.

In another embodiment, hemangioblasts are generated by inducing anypluripotent cell as described herein. In a further embodiment,hemangioblasts are generated by inducing differentiation of apluripotent cell selected from the group comprising blastocysts, platedICMs, one or more blastomeres, or other portions of apre-implantation-stage embryo or embryo-like structure, regardless ofwhether produced by fertilization, somatic cell nuclear transfer (SCNT),parthenogenesis, androgenesis, or other sexual or asexual means, and ESCderived through reprogramming (e.g., iPS cells). In a still furtherembodiment, hemangioblasts are generated from iPS cells, wherein the iPScells are generated using exogenously added factors or other methodsknown in the art such as proteins or microRNA (see Zhou et al., CellStem Cell (4): 1-4, 2009; Miyoshi et al. Cell Stem Cell (8): 1-6, 2011;Danso et al., Cell Stem Cell (8): 376-388, 2011).

In another aspect, the disclosure provides preparations of mesenchymalstromal cells (MSCs) and methods of generating MSCs usinghemangioblasts. The MSC may differ from pre existing MSC in one or moreaspects, as further described herein. In one embodiment, hemangioblastsare harvested after at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 days in culture using a serum free methylcellulose mediumplus one or more ingredients selected from the group comprisingpenicillin/streptomycin (pen/strp), EX-CYTE® growth supplement (awater-soluble concentrate comprising 9.0-11.0 g/L cholesterol and13.0-18.0 g/L lipoproteins and fatty acids at pH 7-8.4), Flt3-ligand(FL), vascular endothelial growth factor (VEGF), thrombopoietin (TPO),basic fibroblast growth factor (bFGF), stem cell derived factor (SCF),granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 3(IL3), and interleukin 6 (IL6), by inducing a pluripotent cell selectedfrom the group comprising blastocysts, plated ICMs, one or moreblastomeres, or other portions of a pre-implantation-stage embryo orembryo-like structure, regardless of whether produced by fertilization,somatic cell nuclear transfer (SCNT), parthenogenesis, androgenesis, orother sexual or asexual means, and cells derived through reprogramming(iPS cells). In a preferred embodiment of the instant invention,hemangioblasts are harvested between 6-14 days, of being cultured in,for example, serum-free methylcellulose plus the ingredients of theprevious embodiment. In a preferred embodiment, the ingredients arepresent in said medium at the following concentrations: Flt3-ligand (FL)at 50 ng/ml, vascular endothelial growth factor (VEGF) at 50 ng/ml,thrombopoietin (TPO) at 50 ng/ml, and basic fibroblast growth factor(bFGF) at 20 ng/ml, 50 ng/ml stem cell derived factor (SCF), 20 ng/mlgranulocyte macrophage colony stimulating factor (GM-CSF), 20 ng/mlinterleukin 3 (IL3), 24 ng/ml interleukin 6 (IL6), 50 ng/ml FL, 50 ng/mlEGF, 50 ng/ml TPO, and 30 ng/ml bFGF.

In another embodiment, a cluster of cells comprised substantially ofhemangioblasts are re-plated and cultured for at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, or 36 days forming a preparation of mesenchymalstromal cells. In one embodiment, mesenchymal stromal cells aregenerated by the steps comprising (a) culturing ESCs for 8-12 days, (b)harvesting hemangioblasts that form clusters of cells, (c) re-platingthe hemangioblasts of step (b), and (d) culturing the hemangioblasts ofstep (c) for between 14-30 days.

In one embodiment, the hemangioblasts are harvested, re-plated andcultured in liquid medium under feeder-free conditions wherein no feederlayer of cells such as mouse embryonic fibroblasts, OP9 cells, or othercell types known to one of ordinary skill in the art are contained inthe culture. In a preferred embodiment, hemangioblasts are cultured onan extracellular matrix. In a further preferred embodiment,hemangioblasts are cultured on an extracellular matrix, wherein saidmatrix comprises a soluble preparation from Engelbreth-Holm-Swarm (EHS)mouse sarcoma cells that gels at room temperature to form areconstituted basement membrane (Matrigel). In a still further preferredembodiment, hemangioblasts are generated according to the stepscomprising (a) culturing said hemangioblasts on Matrigel for at least 7days, (b) transferring the hemangioblasts of step (a) to non-coatedtissue culture plate and further culturing said hemangioblasts of step(b) for between about 7 to 14 days). The hemangioblasts may be culturedon a substrate comprising one or more of the factors selected from thegroup consisting of: transforming growth factor beta (TGF-beta),epidermal growth factor (EGF), insulin-like growth factor 1, bovinefibroblast growth factor (bFGF), and/or platelet-derived growth factor(PDGF), Human Basement Membrane Extract (BME) (e.g., Cultrex BME,Trevigen) or an EHS matrix, laminin, fibronectin, vitronectin,proteoglycan, entactin, collagen (e.g., collagen I, collagen IV), andheparan sulfate. Said matrix or matrix components may be of mammalian,or more specifically human, origin. In one embodiment, hemangioblastsare cultured in a liquid medium comprising serum on a Matrigel-coatedplate, wherein the culture medium may comprise ingredients selected fromαMEM (Sigma-Aldrich) supplemented with 10-20% fetal calf serum (αMEM+20%FCS), αMEM supplemented with 10-20% heat-inactivated human AB serum, andIMDM supplemented with 10-20% heat inactivated AB human serum.

Mesenchymal Stromal Cells Generated by Culturing Hemangioblasts

An embodiment of the instant invention comprises improved mesenchymalstromal cells. The mesenchymal stromal cells of the instant inventionmay be generated from hemangioblasts using improved processes ofculturing hemangioblasts.

Mesenchymal stromal cells of the instant invention may retain higherlevels of potency and may not clump or may clump substantially less thanmesenchymal stromal cells derived directly from ESCs. In an embodimentof the instant invention, a preparation of mesenchymal stromal cellsgenerated according to any one or more of the processes of the instantinvention retains higher levels of potency, and do not clump or clumpsubstantially less than mesenchymal stromal cells derived directly fromESCs.

An embodiment of the instant invention provides a process of culturinghemangioblasts that generate preparations of mesenchymal stromal cells,wherein said mesenchymal stromal cells retain a youthful phenotype. Thepharmaceutical preparations of mesenchymal stromal cells of the instantinvention may demonstrate improved therapeutic properties whenadministered to a mammalian host in need of treatment.

An embodiment of the instant invention provides a preparation ofmesenchymal stromal cells generated by culturing human hemangioblasts. Afurther embodiment of the instant invention provides a process forgenerating a preparation of mesenchymal stromal cells by culturing humanhemangioblasts. In an embodiment of a process of the instant invention,said human hemangioblasts are cultured in feeder-free conditions thenplated on a matrix. In a still further embodiment of the instantinvention, said matrix is selected from the group comprisingtransforming growth factor beta (TGF-beta), epidermal growth factor(EGF), insulin-like growth factor 1, bovine fibroblast growth factor(bFGF), platelet-derived growth factor (PDGF), laminin, fibronectin,vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV,heparan sulfate, a soluble preparation from Engelbreth-Holm-Swarm (EHS)mouse sarcoma cells, Matrigel, and a human basement membrane extract. Ina still further embodiment, said matrix may derive from mammalian orhuman origin.

In another embodiment, hemangioblasts are cultured in a mediumcomprising serum or a serum replacement, such as αMEM supplemented with20% fetal calf serum. In a further embodiment, hemangioblasts arecultured on a matrix for about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In a stillfurther embodiment of the instant invention, a preparation ofmesenchymal stromal cells is generated by the steps comprising (a)culturing hemangioblasts on Matrigel for about 7 days, (b) transferringthe hemangioblasts of step (a) off Matrigel and growing thehemangioblasts on an uncoated tissue culture dish for an additional9-100 days, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 days.

In an embodiment of the instant invention, a preparation of mesenchymalstromal cells is generated by culturing hemangioblasts in a mediumcomprising serum or a serum replacement such as α-MEM supplemented with20% fetal calf serum. In further embodiment of the instant invention,said hemangioblasts are cultured on a matrix for about 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 days.

In an embodiment of the instant invention hemangioblasts aredifferentiated from ESCs. In a further embodiment of the instantinvention, the hemangioblasts of the previous embodiment aredifferentiated from ESCs wherein, said ESCs are selected from the groupcomprising iPS, MA09, H7, H9, MA01, HuES3, H1gfp, NED1, NED2, NED3,NED4, NED5, NED7, inner cell mass cells and blastomere-derived ESCs.

An embodiment of the instant invention comprises a preparation ofmesenchymal stromal cells generated by a process wherein hemangioblastsare differentiated from ESCs. In a further embodiment of the instantinvention, the hemangioblasts of the previous embodiment aredifferentiated from ESCs wherein, said ESCs are selected from the groupcomprising iPS, MA09, H7, H9, MA01, HuES3, H1gfp, inner cell mass cellsand blastomere-derived ESCs.

In an embodiment of the instant invention, hemangioblasts aredifferentiated from ESCs by following the steps comprising: (a)culturing ESCs in the presence of vascular endothelial growth factor(VEGF) and/or bone morphogenic protein 4 (BMP-4) (by way of non-limitingexamples) to form clusters of cells; (b) culturing said clusters ofcells in the presence of at least one growth factor (e.g., basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF), bone morphogenic protein 4 (BMP-4), stem cell factor (SCF), Flt3L (FL), thrombopoietin (TPO), and/or tPTD-HOXB4) in an amountsufficient to induce the differentiation of said clusters of cells intohemangioblasts; and (c) culturing said hemangioblasts in a mediumcomprising at least one additional growth factor (e.g., insulin,transferrin, granulocyte macrophage colony-stimulating factor (GM-CSF),interleukin-3 (IL-3), interleukin-6 (IL-6), granulocytecolony-stimulating factor (G-CSF), erythropoietin (EPO), stem cellfactor (SCF), vascular endothelial growth factor (VEGF), bonemorphogenic protein 4 (BMP-4), and/or tPTD-HOXB4), wherein said at leastone additional growth factor is provided in an amount sufficient toexpand said clusters of cells in said culture, and wherein copper isoptionally added to any of the steps (a)-(c).

In an embodiment of the instant invention, a preparation of mesenchymalstromal cells is generated by culturing hemangioblasts, wherein saidhemangioblasts are differentiated from ESCs by following the stepscomprising: (a) culturing ESCs in the presence of vascular endothelialgrowth factor (VEGF) and bone morphogenic protein 4 (BMP-4) within 0-48hours of initiation of said culture to form clusters of cells; (b)culturing said clusters of cells in the presence of at least one growthfactor selected from the group comprising basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), bone morphogenicprotein 4 (BMP-4), stem cell factor (SCF), Flt 3L (FL), thrombopoietin(TPO), and tPTD-HOXB4 in an amount sufficient to induce thedifferentiation of said clusters of cells into hemangioblasts; and (c)culturing said hemangioblasts in a medium comprising at least oneadditional growth factor selected from the group comprising insulin,transferrin, granulocyte macrophage colony-stimulating factor (GM-CSF),interleukin-3 (IL-3), interleukin-6 (IL-6), granulocytecolony-stimulating factor (G-CSF), erythropoietin (EPO), stem cellfactor (SCF), vascular endothelial growth factor (VEGF), bonemorphogenic protein 4 (BMP-4), and tPTD-HOXB4, wherein said at least oneadditional growth factor is provided in an amount sufficient to expandhuman clusters of cells in said culture.

In another embodiment, a preparation of mesenchymal stem cells isgenerated by the steps comprising: (a) harvesting hemangioblasts afterat least 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of inducing ESCs todifferentiate into said hemangioblasts, and (b) harvesting mesenchymalstromal cells that are generated within about 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 or 50 days of inducing said hemangioblasts from step (a) todifferentiate into said mesenchymal cells.

In yet another embodiment, a preparation of at least 80, 85, 90, 95,100, 125 or 125 million mesenchymal stromal cells are generated fromabout 200,000 hemangioblasts within about 26, 27, 28, 29, 30, 31, 32,33, 34, or 35 days of culturing the hemangioblasts, wherein saidpreparation of mesenchymal stromal cells comprises less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002%, or 0.0001% human embryonic stem cells. In stillanother embodiment, at least 80, 85, 90, 100, 125 or 150 millionmesenchymal stromal cells are generated from about 200,000hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35days of culturing the hemangioblasts.

In an embodiment of a process of the instant invention, a preparation ofmesenchymal stromal cells are substantially purified with respect tohuman embryonic stem cells. In a further embodiment of a process of theinstant invention, a preparation of mesenchymal stromal cells aresubstantially purified with respect to human embryonic stem cells suchthat said preparation comprises at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.

In another embodiment of the instant invention, a preparation ofmesenchymal stromal cells generated by any one or more of the processesof the instant invention does not form a teratoma when introduced into ahost.

In another embodiment of the instant invention, at least 50% of apreparation of mesenchymal stromal cells are positive for CD105 or CD73within about 7-20 (e.g., 15) days of culture. In a preferred embodimentof the instant invention, at least 50% of a preparation of mesenchymalstromal cells generated according to any one or more processes of theinstant invention are positive for CD105 or CD73 after about 7-15 daysof culture. In a further embodiment of the instant invention, at least80% of a preparation of mesenchymal stromal cells are positive for CD105and CD73 within about 20 days of culture. In still a further embodimentof the instant invention, at least 80% of a preparation of mesenchymalstromal cells generated according to any one or more of the processes ofthe instant invention are positive for CD105 and CD73 within about 20days of culture.

In an exemplary aspect, the present disclosure provides a pharmaceuticalpreparation suitable for use in a mammalian patient, comprising at least10⁶ mesenchymal stromal cells and a pharmaceutically acceptable carrier,wherein the mesenchymal stromal cells have replicative capacity toundergo at least 10 population doublings in cell culture with less than25 percent of the cells undergoing cell death, senescing ordifferentiating into non-MSC cells by the tenth doubling.

In order maintain regulatory compliance, MSC banks must maintain asufficient supply of cells, e.g., to provide a sufficient number ofcells to treat at least a few hundred to 10,000 patients, MSC banks musthave at least 50 billion MSCs. The present invention encompassesGMP-complaint and/or cryopreserved MSC banks. In one aspect, the MSCpreparation of the present invention comprise at least 10¹⁰ MSCs such ashemangioblast-derived MSCs. In another aspect, the present inventionprovides a MSC preparation comprising at least 10¹¹, 10¹², 10¹³, or 10¹⁴MSCs such as hemangioblast-derived MSCs.

MSC of the present disclosure, including disclosed compositions,methods, uses, etc., can be derived from a renewable stem source; can bederived from hemgioblast intermediates; and/or may have one or morecharacteristics of the MSC described herein, such as: have, as anaverage, telomere lengths that are at least 30 percent of the telomerelength of an ESC and/or human iPS cell; preparation may, as apopulation, have a mean terminal restriction fragment length (TRF) thatis longer than 4 kb; has a replicative lifespan that is greater than thereplicative lifespan of MSC preparations obtained from other sources(e.g., cultures derived from donated human tissue, such as fetal,infant, child, adolescent or adult tissue); have a statisticallysignificant decreased content and/or enzymatic activity of proteinsinvolved in cell cycle regulation and aging relative to passage 1 (P1),passage 2 (P2), passage 3 (P3), passage 4 (P4) and/or passage 5 (P5) MSCpreparations derived from other sources; have a statisticallysignificant decreased content and/or enzymatic activity of proteinsinvolved in energy and/or lipid metabolism of the cell relative topassage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4) and/orpassage 5 (P5) MSC preparations derived from other sources; have astatistically significant decreased content and/or enzymatic activity ofproteins involved in apoptosis of the cell relative to passage 1 (P1),passage 2 (P2), passage 3 (P3), passage 4 (P4) and/or passage 5 (P5) MSCpreparations derived from other sources; and the MSCs present in thetherapeutic dose (and bank from which it is drawn) are a homogeneouspopulation with respect to which MI-IC genes/alleles are expressed(i.e., are MHC matched within the dose of cells).

In an exemplary aspect, the present disclosure provides a pharmaceuticalpreparation suitable for use in a mammalian patient comprising at least10⁶ mesenchymal stromal cells and a pharmaceutically acceptable carrier,wherein the mesenchymal stromal cells have replicative capacity toundergo at least 5 passages in cell culture with less than 25 percent ofthe cells undergoing cell death, senescing or differentiating intofibroblasts by the 5^(th) passage.

In an exemplary aspect, the present disclosure provides a pharmaceuticalpreparation comprising at least 10⁶ mesenchymal stromal cells and apharmaceutically acceptable carrier, wherein the mesenchymal stromalcells are differentiated from hemangioblasts.

In an exemplary aspect, the present disclosure provides a cryogenic cellbank comprising at least 10⁸ mesenchymal stromal cells, wherein themesenchymal stromal cells have replicative capacity to undergo at least10 population doublings in cell culture with less than 25 percent of thecells undergoing cell death, senescing or differentiating intofibroblasts by the tenth population doubling.

In an exemplary aspect, the present disclosure provides a purifiedcellular preparation comprising at least 10⁶ mesenchymal stromal cellsand less than one percent of any other cell type, wherein themesenchymal stromal cells have replicative capacity to undergo at least10 population doublings in cell culture with less than 25 percent of thecells undergoing cell death, senescing or differentiating into non-MSCcells by the tenth population doubling.

The mesenchymal stromal cells may be differentiated from a pluripotentstem cell source, such as an embryonic stem cell line or inducedpluripotent stem cell line. For example, all of the mesenchymal stromalcells of the preparation or bank may be differentiated from a commonpluripotent stem cell source. Additionally, the mesenchymal stromalcells may be differentiated from a pluripotent stem cell source,passaged in culture to expand the number of mesenchymal stromal cells,and isolated from culture after less than twenty population doublings.

The mesenchymal stromal cells may be HLA-genotypically identical. Themesenchymal stromal cells may be genomically identical.

At least 30% of the mesenchymal stromal cells may be positive for CD10.Additionally, at least 60% of the mesenchymal stromal cells may bepositive for markers CD73, CD90, CD105, CD13, CD29, CD44, and CD166 andHLA-ABC. In an exemplary embodiment, less than 30% of the mesenchymalstromal cells may be positive for markers CD31, CD34, CD45, CD133,FGFR2, CD271, Stro-1, CXCR4 and TLR3.

The mesenchymal stromal cells may have replicative rates to undergo atleast 10 population doublings in cell culture in less than 25 days. Themesenchymal stromal cells may have a mean terminal restriction fragmentlength (TRF) that may be longer than 8 kb. The mesenchymal stromal cellsmay have a statistically significant decreased content and/or enzymaticactivity, relative to mesenchymal stromal cell preparations derived frombone marrow that have undergone five population doublings, of proteinsinvolved in one or more of (i) cell cycle regulation and cellular aging,(ii) cellular energy and/or lipid metabolism, and (iii) apoptosis. Themesenchymal stromal cells may have a statistically significant increasedcontent and/or enzymatic activity of proteins involved in cytoskeletonstructure and cellular dynamics relating thereto, relative tomesenchymal stromal cell preparations derived from bone marrow. Themesenchymal stromal cells may not undergo more than a 75 percentincrease in cells having a forward-scattered light value, measured byflow cytometry, greater than 5,000,000 over 10 population doublings inculture. The mesenchymal stromal cells may, in a resting state, expressmRNA encoding Interleukin-6 at a level which may be less than tenpercent of the IL-6 mRNA level expressed by mesenchymal stromal cellspreparations, in a resting state, derived from bone marrow or adiposetissue.

The preparation may be suitable for administration to a human patient.The preparation may be suitable for administration to a non-humanveterinarian mammal.

In an exemplary aspect, the disclosure provides a pharmaceuticalpreparation comprising mesenchymal stromal cells, wherein saidmesenchymal stromal cells are able to undergo at least 10 populationdoublings and wherein the 10 population doublings occur within about 27days, more preferably less than about 26 days, preferably less than 25days, more preferably less than about 24 days, still more preferablyless than about 23 days, still more preferably less than about 22 days,or lower.

In an exemplary aspect, the disclosure provides a pharmaceuticalpreparation comprising mesenchymal stromal cells, wherein saidmesenchymal stromal cells are able to undergo at least 15 populationdoublings.

Said mesenchymal stromal cells may be able to undergo at least 20, 25,30, 35, 40, 45, 50 or more population doublings.

In an exemplary aspect, the disclosure provides a pharmaceuticalpreparation comprising mesenchymal stromal cells, wherein saidmesenchymal stromal cells are able to undergo at least 15 populationdoublings, at least 20 population doublings, or at least 25 populationdoublings in culture.

The mesenchymal stromal cells may be produced by in vitrodifferentiation of hemangioblasts. The mesenchymal stromal cells may beprimate cells or other mammalian cells. The mesenchymal stromal cellsmay be human cells.

Said population doublings occur within about 35 days, more preferablywithin about 34 days, preferably within 33 days, more preferably within32 days, still more preferably within 31 days, or still more preferablywithin about 30 days.

The preparation may comprise less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or0.0001% pluripotent cells.

The preparation may be devoid of pluripotent cells.

The preparation may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% mesenchymal stromal cells.

At least 50% of said mesenchymal stromal cells may be positive for (i)at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 andCD90; (ii) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1,CD105, CD73, CD90, CD105, CD13, CD29, CD 44, CD166, CD274, and HLA-ABC;(iii) CD105, CD73 and/or CD90 or (iv) any combination thereof. At least50% of said mesenchymal stromal cells may be positive for (i) at leasttwo of CD105, CD73 and/or CD90 (ii) at least two of CD10, CD24, IL-11,AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (iii) all of CD10, CD24,IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29, CD44,CD166, CD274, and HLA-ABC. At least 50% of said mesenchymal stromalcells (i) may be positive for all of CD105, CD73 and CD90; (ii) positivefor all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90,CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC and/or (ii) may benegative for or less than 5% or less than 10% of the cells express CD31,34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, and/or TLR3. At least 60%,70%, 80% or 90% of said mesenchymal stromal cells may be positive for(i) one or more of CD105, CD73 and CD90 (ii) one or more of CD10, CD24,IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (iii) one or moreof CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105,CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

The pharmaceutical preparation may comprise an amount of mesenchymalstromal cells effective to treat an unwanted immune response in asubject in need thereof.

The pharmaceutical preparation may comprise other cells, tissue or organfor transplantation into a recipient in need thereof. The other cells ortissue may be RPE cells, skin cells, corneal cells, pancreatic cells,liver cells, cardiac cells or tissue containing any of said cells. Saidmesenchymal stromal cells may be not derived from bone marrow and thepotency of the preparation in an immune regulatory assay may be greaterthan the potency of a preparation of bone marrow derived mesenchymalstromal cells. Potency may be assayed by an immune regulatory assay thatdetermines the EC50 dose. The preparation may retain between about 50and 100% of its proliferative capacity after ten population doublings.

Said mesenchymal stromal cells may be not derived directly frompluripotent cells and wherein said mesenchymal stromal cells (a) do notclump or clump substantially less than mesenchymal stromal cells deriveddirectly from pluripotent cells; (b) more easily disperse when splittingcompared to mesenchymal stromal cells derived directly from pluripotentcells; (c) may be greater in number than mesenchymal stromal cellsderived directly from pluripotent cells when starting with equivalentnumbers of pluripotent cells; and/or (d) acquire characteristicmesenchymal cell surface markers earlier than mesenchymal stromal cellsderived directly from pluripotent cells.

Said mesenchymal stromal cells may be mammalian. Said mesenchymalstromal cells may be human, canine, bovine, non-human primate, murine,feline, or equine

In an exemplary aspect, the present disclosure provides a method forgenerating mesenchymal stromal cells comprising culturing hemangioblastsunder conditions that give rise to mesenchymal stem cells. Saidhemangioblasts may be cultured in feeder-free conditions. Saidhemangioblasts may be plated on a matrix. Said matrix may comprise oneor more of: transforming growth factor beta (TGF-beta), epidermal growthfactor (EGF), insulin-like growth factor 1, bovine fibroblast growthfactor (bFGF), and/or platelet-derived growth factor (PDGF). Said matrixmay be selected from the group consisting of: laminin, fibronectin,vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV,heparan sulfate, Matrigel (a soluble preparation fromEngelbreth-Holm-Swann (EHS) mouse sarcoma cells), a human basementmembrane extract, and any combination thereof. Said matrix may comprisea soluble preparation from Engelbreth-Holm-Swann mouse sarcoma cells.

Said mesenchymal stromal cells may be mammalian. Said mesenchymalstromal cells may be human, canine, bovine, non-human primate, murine,feline, or equine.

Said hemangioblasts may be cultured in a medium comprising αMEM. Saidhemangioblasts may be cultured in a medium comprising serum or a serumreplacement. Said hemangioblasts may be cultured in a medium comprising,αMEM supplemented with 0%, 0.1% 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% fetal calfserum. Said hemangioblasts may be cultured on said matrix for at leastabout 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 days.

Said hemangioblasts may be differentiated from pluripotent cells.

Said pluripotent cells may be iPS cells or pluripotent cells producedfrom blastomeres. Said pluripotent cells may be derived from one or moreblastomeres without the destruction of a human embryo.

Said hemangioblasts may be differentiated from pluripotent cells by amethod comprising (a) culturing said pluripotent cells to form clustersof cells. The pluripotent cells may be cultured in the presence ofvascular endothelial growth factor (VEGF) and/or bone morphogenicprotein 4 (BMP-4). In step (a), the pluripotent cells may be cultured inthe presence of vascular endothelial growth factor (VEGF) and/or bonemorphogenic protein 4 (BMP-4). Said VEGF and BMP-4 may be added to thepluripotent cell culture within 0-48 hours of initiation of said cellculture, and said VEGF may be optionally added at a concentration of20-100 nm/mL and said BMP-4 may be optionally added at a concentrationof 15-100 ng/mL. Said VEGF and BMP-4 may be added to the cell culture ofstep (a) within 0-48 hours of initiation of said cell culture, and saidVEGF may be optionally added at a concentration of 20-100 nm/mL and saidBMP-4 may be optionally added at a concentration of 15-100 ng/mL. Saidhemangioblasts may be differentiated from pluripotent cells by a methodwhich may further comprise: (b) culturing said clusters of cells in thepresence of at least one growth factor in an amount sufficient to inducethe differentiation of said clusters of cells into hemangioblasts. Saidat least one growth factor added in step (b) may comprise one or more ofbasic fibroblast growth factor (bFGF), vascular endothelial growthfactor (VEGF), bone morphogenic protein 4 (BMP-4), stem cell factor(SCF), Flt 3L (FL), thrombopoietin (TPO), EPO, and/or tPTD-HOXB4.

Said at least one growth factor added in step (b) may comprise one ormore of: about 20-25 ng/ml basic fibroblast growth factor (bFGF), about20-100 ng/ml vascular endothelial growth factor (VEGF), about 15-100ng/ml bone morphogenic protein 4 (BMP-4), about 20-50 ng/ml stem cellfactor (SCF), about 10-50 ng/ml Fit 3L (FL), about 20-50 ng/mlthrombopoietin (TPO), EPO, and/or 1.5-5 U/ml tPTD-HOXB4.

One or more of said at least one growth factor optionally added in step(b) may be added to said culture within 36-60 hours or 40-48 hours fromthe start of step (a).

One or more of said at least one growth factor added in step (b) may beadded to said culture within 48-72 hours from the start of step (a).

Said at least one factor added in step (b) may comprise one or more ofbFGF, VEGF, BMP-4, SCF and/or FL.

The method may further comprise (c) dissociating said clusters of cells,optionally into single cells.

The method may further comprise (d) culturing said hemangioblasts in amedium comprising at least one additional growth factor, wherein said atleast one additional growth factor may be in an amount sufficient toexpand the hemangioblasts.

In step (d), said at least one additional growth factor may comprise oneor more of: insulin, transferrin, granulocyte macrophagecolony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6(IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin(EPO), stem cell factor (SCF), vascular endothelial growth factor(VEGF), bone morphogenic protein 4 (BMP-4), and/or tPTD-HOXB4.

In step (d), said at least one additional growth factor may comprise oneor more of: about 10-100 μg/ml insulin, about 200-2,000 μg/mltransferrin, about 10-50 ng/ml granulocyte macrophage colony-stimulatingfactor (GM-CSF), about 10-20 ng/ml interleukin-3 (IL-3), about 10-1000ng/ml interleukin-6 (IL-6), about 10-50 ng/ml granulocytecolony-stimulating factor (G-CSF), about 3-50 U/ml erythropoietin (EPO),about 20-200 ng/ml stem cell factor (SCF), about 20-200 ng/ml vascularendothelial growth factor (VEGF), about 15-150 ng/ml bone morphogenicprotein 4 (BMP-4), and/or about 1.5-15 U/ml tPTD-HOXB4.

Said medium in step (a), (b), (c) and/or (d) may be a serum-free medium.

The method as described above may further comprise (e) mitoticallyinactivating the mesenchymal stromal cells.

At least 80, 85, 90, 95, 100, 125, or 150 million mesenchymal stromalcells may be generated.

Said hemangioblasts may be harvested after at least 10, 11, 12, 13, 14,15, 16, 17 or 18 days of starting to induce differentiation of saidpluripotent cells.

Said mesenchymal stromal cells may be generated within at least 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 days of starting to induce differentiation ofsaid pluripotent cells.

The method may result in at least 80, 85, 90, 95, 100, 125, or 150million mesenchymal stromal cells being generated from about 200,000hemangioblasts within about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of culture.

The mesenchymal stromal cells may be generated from hemangioblasts in aratio of hemangioblasts to mesenchymal stromal cells of at least 1:200,1:250, 1:300, 1:350, 1:400, 1:415, 1:425, 1:440; 1:450, 1:365, 1:475,1:490 and 1:500 within about 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35days of culture as hemangioblasts.

Said cells may be human.

In another aspect, the present disclosure provides mesenchymal stromalcells derived from hemangioblasts obtained by any of the methodsdescribed above.

In another aspect, the present disclosure provides mesenchymal stromalcells derived by in vitro differentiation of hemangioblasts.

At least 50% of said mesenchymal stromal cells (i) may be positive forall of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90,CD105, CD13, CD29, CD44, CD166, CD274, and HLA-ABC and (ii) may benegative for or less than 5% or less than 10% of the cells express CD31,34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and/or TLR3.

At least 50% of said mesenchymal stromal cells may be positive for (i)all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or(ii) all of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, andHLA-ABC.

At least 60%, 70%, 80% or 90% of said mesenchymal stromal cells may bepositive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1,CXCL1, CD105, CD73 and CD90; or (ii) at least one of CD73, CD90, CD105,CD13, CD29, CD44, CD166, CD274, and HLA-ABC.

The mesenchymal stromal may not express or less than 5% or less than 10%of the cells may express at least one of CD31, 34, 45, 133, FGFR2,CD271, Stro-1, CXCR4, or TLR3.

In another aspect, the present disclosure provides a preparation ofmesenchymal stromal cells as described above.

Said preparation may comprise less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or0.0001% pluripotent cells.

The preparation may be devoid of pluripotent cells.

Said preparation may be substantially purified and optionally maycomprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% human mesenchymal stromal cells.

The preparation may comprise substantially similar levels of p53 and p21protein or wherein the levels of p53 protein as compared to p21 proteinmay be 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater.

The mesenchymal stromal cells or the MSC in the preparation may becapable of undergoing at least 5 population doublings in culture, or maybe capable of undergoing at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60 or more population doublings in culture.

Said mesenchymal stromal cells: (a) may not clump or clump substantiallyless than mesenchymal stromal cells derived directly from pluripotentcells; (b) may more easily disperse when splitting compared tomesenchymal stromal cells derived directly from pluripotent cells; (c)may be greater in number than mesenchymal stromal cells derived directlyfrom pluripotent cells when starting with equivalent numbers ofpluripotent cells; and/or (d) may acquire characteristic mesenchymalcell surface markers earlier than mesenchymal stromal cells deriveddirectly from pluripotent cells.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising any mesenchymal stromal cells or preparation of mesenchymalstromal cells as described above.

The pharmaceutical preparation may comprise an amount of mesenchymalstromal cells effective to treat an unwanted immune response.

The pharmaceutical preparation may comprise an amount of mesenchymalstromal cells effective to treat an unwanted immune response and mayfurther comprise other cells or tissues for transplantation into arecipient in need thereof.

Said other cells or tissues may be allogeneic or syngeneic pancreatic,neural, liver, RPE, corneal cells or tissues containing any of theforegoing cells.

The pharmaceutical preparation may be for use in treating an autoimmunedisorder or an immune reaction against allogeneic cells, or for use intreating multiple sclerosis, systemic sclerosis, hematologicalmalignancies, myocardial infarction, organ transplantation rejection,chronic allograft nephropathy, cirrhosis, liver failure, heart failure,GvHD, tibial fracture, bone factures, left ventricular dysfunction,leukemia, myelodysplastic syndrome, Crohn's disease, diabetes, obesity,metabolic diseases and disorders (such as lysosomal storage diseasesincluding Tay-Sachs disease, Gaucher disease, Pompe disease, Hurlersyndrome and metachromatic leukodystrophy), fatty liver disease, chronicobstructive pulmonary disease, osteogenesis imperfecta, homozygousfamilial hypocholesterolemia, hypocholesterolemia, treatment followingmeniscectomy, adult periodontitis, periodontitis, vasculogenesis inpatients with severe myocardial ischemia, spinal cord injury,osteodysplasia, critical limb ischemia, diabetic foot disease, primarySjogren's syndrome, osteoarthritis, cartilage defects, laminitis,multisystem atrophy, amyotropic lateral sclerosis, cardiac surgery,systemic lupus erythematosis, living kidney allografts, nonmalignant redblood cell disorders, thermal burn, radiation burn, Parkinson's disease,microfractures, epidermolysis bullosa, severe coronary ischemia,idiopathic dilated cardiomyopathy, osteonecrosis femoral head, lupusnephritis, bone void defects, ischemic cerebral stroke, after stroke,acute radiation syndrome, pulmonary disease, arthritis, boneregeneration, uveitis or combinations thereof.

In another aspect, the disclosure provides a kit comprising any of themesenchymal stromal cells or any preparation of mesenchymal stromalcells as described above.

In another aspect, the disclosure provides a kit comprising themesenchymal stromal cells or preparation of mesenchymal stromal cells asdescribed above, wherein said cells or preparation of cells may befrozen or cryopreserved.

In another aspect, the disclosure provides a kit comprising themesenchymal stromal cells or preparation of mesenchymal stromal cells asdescribed above, wherein said cells or preparation of cells may becontained in a cell delivery vehicle.

In another aspect, the disclosure provides a method for treating adisease or disorder, comprising administering an effective amount ofmesenchymal stromal cells or a preparation of mesenchymal stromal cellsas described above to a subject in need thereof.

The method may further comprise the transplantation of other cells ortissues. The cells or tissues may comprise retinal, RPE, corneal,neural, immune, bone marrow, kidney, liver or pancreatic cells. Thedisease or disorder may be selected from multiple sclerosis, systemicsclerosis, hematological malignancies, myocardial infarction, organtransplantation rejection, chronic allograft nephropathy, cirrhosis,liver failure, heart failure, GvHD, tibial fracture, bone factures, leftventricular dysfunction, leukemia, myelodysplastic syndrome, Crohn'sdisease, diabetes, obesity, metabolic diseases and disorders (such aslysosomal storage diseases including Tay-Sachs disease, Gaucher disease,Pompe disease, Hurler syndrome and metachromatic leukodystrophy), fattyliver disease, chronic obstructive pulmonary disease, osteogenesisimperfecta, homozygous familial hypocholesterolemia,hypocholesterolemia, treatment following meniscectomy, adultperiodontitis, periodontitis, vasculogenesis in patients with severemyocardial ischemia, spinal cord injury, osteodysplasia, critical limbischemia, diabetic foot disease, primary Sjogren's syndrome,osteoarthritis, cartilage defects, multisystem atrophy, amyotropiclateral sclerosis, cardiac surgery, refractory systemic lupuserythematosis, living kidney allografts, nonmalignant red blood celldisorders, thermal burn, Parkinson's disease, microfractures,epidermolysis bullosa, severe coronary ischemia, idiopathic dilatedcardiomyopathy, osteonecrosis femoral head, lupus nephritis, bone voiddefects, ischemic cerebral stroke, after stroke, acute radiationsyndrome, pulmonary disease, arthritis, bone regeneration, orcombinations thereof.

The disease or disorder may be uveitis. Said disease or disorder may bean autoimmune disorder or an immune reaction against allogeneic cells.The autoimmune disorder may be multiple sclerosis.

In another aspect, the disclosure provides a method of treating boneloss or cartilage damage comprising administering an effective amount ofmesenchymal stromal cells or preparation of mesenchymal stromal cells toa subject in need thereof.

The mesenchymal stromal cells may be administered in combination with anallogeneic or syngeneic transplanted cell or tissue. The allogeneictransplanted cell may comprise a retinal pigment epithelium cell,retinal cell, corneal cell, or muscle cell.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising mitotically inactivated mesenchymal stromal cells. Themesenchymal stromal cells may be differentiated from a hemangioblastcell.

The pharmaceutical may comprise at least 10⁶ mesenchymal stromal cellsand a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising mitotically inactivated mesenchymal stromal cells produced bythe method above.

The preparation may be suitable for administration to a human patient.The preparation may be suitable for administration to a non-humanveterinarian mammal.

The pharmaceutical preparation may be devoid of pluripotent cells.

The pharmaceutical preparation may comprise an amount of mesenchymalstromal cells effective to treat an unwanted immune response in asubject in need thereof.

The pharmaceutical preparation may comprise an amount of mesenchymalstromal cells effective to treat a disease or condition selected fromthe group consisting of: inflammatory respiratory conditions,respiratory conditions due to an acute injury, Adult RespiratoryDistress Syndrome, post-traumatic Adult Respiratory Distress Syndrome,transplant lung disease, Chronic Obstructive Pulmonary Disease,emphysema, chronic obstructive bronchitis, bronchitis, an allergicreaction, damage due to bacterial pneumonia, damage due to viralpneumonia, asthma, exposure to irritants, tobacco use, atopicdermatitis, allergic rhinitis, hearing loss, autoimmune hearing loss,noise-induced hearing loss, psoriasis and any combination thereof.

Preparation of Mesenchymal Stromal Cells

In an embodiment of the instant invention, a preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts)is provided, wherein the desired phenotype of said mesenchymal stromalcells presents earlier as compared to mesenchymal stromal cells by ESCculture (See FIG. 5). In a further embodiment of the instant invention,a preparation of the subject mesenchymal stromal cells (e.g., generatedby culturing hemangioblasts) is provided, wherein the desired phenotypeof said mesenchymal stromal cells presents earlier as compared tomesenchymal stromal cells by ESC culture, and wherein said desiredphenotype is defined by the expression of at least two markers selectedfrom the group comprising CD9, CD13, CD29, CD44, CD73, CD90, CD105,CD166, and HLA-ABC.

A further embodiment of the instant invention comprises a preparation ofmesenchymal stromal cells, wherein the phenotype of said mesenchymalstromal cells is defined by the expression of at least two markersselected from the group comprising CD9, CD13, CD29, CD44, CD73, CD90,CD105, CD166, and HLA-ABC. A still further embodiment of the instantinvention comprises a preparation of mesenchymal stromal cells, whereinthe phenotype of said mesenchymal stromal cells is defined by theexpression of at least two markers selected from the group comprisingCD9, CD13, CD29, CD44, CD73, CD90 and CD105, and wherein saidmesenchymal stromal cells do not express CD2, CD3, CD4, CD5, CD7, CD8,CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E andCD133.

In an embodiment of the instant invention about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the subject mesenchymalstromal cells (e.g., generated by culturing hemangioblasts) present aphenotype defined by the expression of the markers CD9, CD13, CD29,CD44, CD73, CD90, CD105, CD166, and HLA-abc after about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in culture. In anembodiment of the instant invention at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the subject mesenchymalstromal cells (e.g., generated by culturing hemangioblasts) present aphenotype defined by the expression of at least two markers selectedfrom the group comprising CD9, CD13, CD29, CD44, CD73, CD90, CD105,CD166, and HLA-ABC and a lack of expression of CD2, CD3, CD4, CD5, CD7,CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E,CD133 and Stro-1 after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 days in culture. The previous embodiment, whereinsaid phenotype is further defined by the markers selected from the groupcomprising AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

A preferred process of the instant invention is provided, wherein thenumber of mesenchymal stromal cells derived from hemangioblasts is about8×10⁷, 8.5×10⁷, 9×10⁷, 9.5×10⁷, 1×10⁸, 1.25×10⁸, or 1.5×10⁸ mesenchymalstromal cells derived from about 2×10⁵ hemangioblasts within about 30days of culture of mesenchymal stromal cells. In an alternativeembodiment of the instant invention, mesenchymal stromal cells may begenerated from hemangioblasts in a ratio of hemangioblasts tomesenchymal stromal cells of about 1:200, 1:400, 1:415, 1:425, 1:440;1:450, 1:465, 1:475, 1:490, and 1:500, within about 30 days of cultureof mesenchymal stromal cells.

In a preferred embodiment of the instant invention, the number ofmesenchymal stromal cells obtained by hemangioblast culture is higherthan the number of mesenchymal stromal cells obtained directly fromESCs. In a further preferred embodiment of the instant invention, thenumber of mesenchymal stromal cells obtained by hemangioblast culture isat least 5 times, 10 times, 20 times, 22 times higher than the number ofmesenchymal stromal cells obtained directly from ESCs than the number ofmesenchymal stromal cells obtained directly from ESCs (See FIG. 4).

In another embodiment of the instant invention, a preparation of thesubject mesenchymal stromal cells does not form teratomas whenintroduced into mammalian host.

An embodiment of the instant invention provides a preparation ofmesenchymal stromal cells generated by culturing hemangioblasts usingany of the process embodiments of the instant invention. An embodimentof the instant invention comprising a preparation of mesenchymal stromalcells is generated by culturing hemangioblasts using any of the processembodiments of the instant invention, wherein the phenotype of saidpreparation is defined by the presence of any or all of the markersselected from the group comprising AIRE-1, IL-11, CD10, CD24, ANG-1, andCXCL1. A further embodiment of the instant invention comprising apreparation of mesenchymal stromal cells is generated by culturinghemangioblasts using any of the process embodiments of the instantinvention, wherein the phenotype of said preparation is defined by thepresence of any or all of the markers selected from the group comprisingAIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1, and wherein saidpreparation presents a reduced expression of IL-6, Stro-1 and VEGF.

In an embodiment of the instant invention, a preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts)is provided, wherein said preparation comprises substantially similarlevels of p53 and p21 protein, or wherein the levels of p53 as comparedto p21 are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In anembodiment of the instant invention, a pharmaceutical preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) is provided, wherein said pharmaceutical preparationcomprises substantially similar levels of p53 and p21 protein, orwherein the levels of p53 as compared to p21 are 1.5, 2, 3, 4, 5, 6, 7,8, 9, or 10 times greater.

In an embodiment of the instant invention, a preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts)is provided, wherein said preparation comprises a substantially similarpercentage of cells positive for p53 and p21 protein, or wherein thepercentage of cells positive for p53 as compared to p21 are 1.5, 2, 3,4, 5, 6, 7, 8, 9, or 10 times greater. In an embodiment of the instantinvention, a preparation of the subject mesenchymal stromal cells (e.g.,generated by culturing hemangioblasts) is provided wherein saidpreparation comprises a substantially similar percentage of cellspositive for p53 and p21 protein, or wherein the percentage of cellspositive for p53 as compared to p21 are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or10 times greater. In an embodiment of the instant invention, apharmaceutical preparation of the subject mesenchymal stromal cells(e.g., generated by culturing hemangioblasts) is provided, wherein saidpharmaceutical preparation comprises a substantially similar percentageof cells positive for p53 and p21 protein, or wherein the percentage ofcells positive for p53 as compared to p21 are 1.5, 2, 3, 4, 5, 6, 7, 8,9, or 10 times greater.

In an embodiment of the instant invention, a preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts)is provided, wherein said preparation comprises a substantially similarpercentage of cells having background levels of aging markers selectedfrom the group comprising S100A1, VIM, MYADM, PIM1, ANXA2, RAMP, MEG3,IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B, LASS6, ROBO1,DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, SLC35E1, ANXA2,HLA-E, CD59, BHLHB2, UCHL1, SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B, IDS,TGFBR2, TSPAN6, TM4SF1, MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A, VAMP1,ADD1, FAM129A, HPDC1, KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2,KIAA1191, C5ORF32, C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41,DUSP18, SH3PX3, MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103,EPOR, and TMEM112 or from the group comprising S100A1, VIM, MYADM, PIM1,ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EMLI, MYO1B,LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, andSLC35E1, or wherein the percentage of cells positive for aging markersselected from the group comprising S100A1, VIM, MYADM, PIM, ANXA2, RAMP,MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B, LASS6,ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, SLC35E1,ANXA2, HLA-E, CD59, BHLHB2, UCHL1, SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B,IDS, TGFBR2, TSPAN6, TM4SF1, MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A, VAMP1ADD1, FAM129A, HPDC1, KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2,KIAA1191, C5ORF32, C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41,DUSP18, SH3PX3, MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103,EPOR, TMEM112 or from the group comprising S100A1, VIM, MYADM, PIM1,ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1, DGKA, TPBG, MGLL, EML1, MYO1B,LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, andSLC35E1, are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater thanbackground. In an embodiment of the instant invention, a preparation ofthe subject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) is provided, wherein said preparation comprises asubstantially similar percentage of cells having background levels ofmarkers selected from the group comprising HoxB3, HoxB7, MID1, SNAPC5,PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1CRIP1, SULT1A3, STMN1, CCT8,SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2,CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10,TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A,GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5,ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C160RF63, FAM33A,EPSTL1, CTR9, GAS5, ZNF711, MTO1, and CDP2, or wherein the percentage ofcells positive for markers selected from the group comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1,SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ111021, DDX46, ACADM,KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR,EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02,SHMT2, ACP1, FKBP3, ARL5A, GMNN, ENY2, FAM82B, RNF138, RPL26L1, CCDC59,PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1,MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1, and CDP2 are1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times less than background.

In an embodiment of the instant invention, a preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts)is provided wherein said preparation comprises a substantially similarpercentage of cells having background levels of aging markers selectedfrom the group comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2,TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3,CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6,CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A,LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A, GMNN, ENY2,FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47,GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9,GAS5, ZNF711, MTO1, and CDP2, or from the group comprising HoxB3, HoxB7,MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1 and SULT1A3or wherein the percentage of cells positive for aging markers selectedfrom the group comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2,TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3,CBX1, FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6,CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A,LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A, GMNN, ENY2,FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47,GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9,GAS5, ZNF711, MTO1, and CDP2 or the group comprising HoxB3, HoxB7, MID1,SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3 are 1.5,2, 3, 4, 5, 6, 7, 8, 9, or 10 times less than background.

In another embodiment, the MSCs (such as hemangioblast-derived MSCs)possess phenotypes of younger cells as compared to adult-derived MSCs.In one embodiment, the subject MSCs are capable of undergoing at leastor about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or morepopulation doublings in culture. In contrast, adult-derived mesenchymalstromal cells typically undergo 2-3 doublings in culture. In anotherembodiment, the MSCs (such as hemangioblast-derived MSCs) have longertelomere lengths, greater immunosuppressive effects, fewer vacuoles,divide faster, divide more readily in culture, higher CD90 expression,are less lineage committed, or combinations thereof, compared toadult-derived MSCs. In another embodiment, the hemangioblast-derived MSChave increased expression of transcripts promoting cell proliferation(i.e., have a higher proliferative capacity) and reduced expression oftranscripts involved in terminal cell differentiation compared toadult-derived MSCs.

In an embodiment of the instant invention, a preparation of mesenchymalstromal cells is generated by any one or more of the processes of theinstant invention, wherein said mesenchymal stromal cells are capable ofundergoing at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, or more population doublings in culture.

In another embodiment of the instant invention, a preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) are capable of undergoing at least or about 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, or more population doublings inculture. In another embodiment of the instant invention, a preparationof the subject mesenchymal stromal cells are capable of undergoing atleast or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or morepopulation doublings in culture, wherein after said population doublingsless than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of mesenchymal stromalcells have undergone replicative senescence. In a further embodiment,said preparation is a pharmaceutical preparation.

In another embodiment of the instant invention, a preparation ofmesenchymal stromal cells is provided, wherein said mesenchymal stromalcells have undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 doublings in culture,

In another embodiment of the instant invention, a preparation ofmesenchymal stromal cells is provided, wherein said mesenchymal stromalcells have undergone at least or about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 doublings in culture, wherein less than 50%, 40%, 30%,20%, 15%, 10%, 5%, or 1% of said mesenchymal stromal cells haveundergone replicative senescence, wherein said mesenchymal stromal cellsretain a youthful phenotype and potency, and wherein said preparation isa pharmaceutical preparation. Said preparation may comprise an effectivenumber of mesenchymal stromal cells for the treatment of disease, suchas an immunological disorder, degenerative disease, or other diseaseamenable to treatment using MSCs.

In another embodiment of the instant invention, a preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) is provided, wherein said mesenchymal stromal cells haveundergone at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,or 60 doublings in culture, wherein less than 50%, 40%, 30%, 20%, 15%,10%, 5%, or 1% of said mesenchymal stromal cells have undergonereplicative senescence after such doublings, wherein said mesenchymalstromal cells retain a youthful phenotype and potency, and wherein saidpreparation is a pharmaceutical preparation. Said preparation maycomprise an effective number of mesenchymal stromal cells for thetreatment of disease, such as an immunological disorder, degenerativedisease, or other disease amenable to treatment using MSCs.

In another embodiment of the instant invention, a preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) is provided, wherein said mesenchymal stromal cells haveundergone about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60doublings in culture. The previous embodiment wherein less than 50%,40%, 30%, 20%, 15%, 10%, 5% 1% of said mesenchymal stromal cells haveundergone replicative senescence, wherein said mesenchymal stromal cellsretain a youthful phenotype and potency, wherein said preparation is apharmaceutical preparation, wherein said pharmaceutical preparationcomprises an effective number of mesenchymal stromal cells, and whereinsaid pharmaceutical preparation is preserved.

In another embodiment, the instant invention provides a kit comprising apharmaceutical preparation of mesenchymal stromal cells. In anotherembodiment, the instant invention provides a kit comprising apharmaceutical preparation of mesenchymal stromal cells, wherein saidpreparation is preserved. In another embodiment, the instant inventionprovides a kit comprising a pharmaceutical preparation of the subjectmesenchymal stromal cells (e.g., generated by culturing hemangioblasts).In another embodiment, the instant invention provides a kit comprising apharmaceutical preparation of the subject mesenchymal stromal cells(e.g., generated by culturing hemangioblasts), wherein said preparationis preserved.

In another embodiment, the instant invention provides for a method oftreating a pathology by administering an effective amount of mesenchymalstromal cells derived from hemangioblasts to a subject in need thereof.Said pathology may include, but is not limited to an autoimmunedisorder, uveitis, bone loss or cartilage damage.

The mesenchymal stromal cells obtained by culturing hemangioblasts haveimproved characteristics as compared to MSCs derived directly from ESCs.For example, ESC-derived MSCs clump more, are more difficult to dispersewhen splitting, do not generate nearly as many MSCs when starting withequivalent numbers of ESCs, and take longer to acquire characteristicsMSC cell surface markers compared to hemangioblast-derived MSCs. SeeExample 2 and FIGS. 3-6.

In one embodiment, the instant invention provides a preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts), wherein said preparation is effective at normalizing apathology. In a further embodiment of the instant invention apreparation of the subject mesenchymal stromal cells (e.g., generated byculturing hemangioblasts) is provided, wherein said preparation iseffective at reducing excessive or unwanted immune responses. In afurther embodiment of the instant invention, a preparation of thesubject mesenchymal stromal cells (e.g., generated by culturinghemangioblasts) is provided, wherein said preparation is effective atameliorating an autoimmune disorder. In a further embodiment of theinstant invention, normalization of a pathology by administering to ahost an effective amount of the subject mesenchymal stromal cells (e.g.,generated by culturing hemangioblasts) is provided. A further embodimentof the instant invention provides for normalization of a pathology,wherein such normalization of a pathology is characterized by effectsselected from the group comprising cytokine release by said MSCs,stimulating an increase in the number of regulatory T cells, inhibitinga certain amount of IFN gamma release from Th1 cells, and stimulating acertain amount of IL4 secretion from Th2 cells. In a further embodiment,administration of a preparation of the subject mesenchymal stromal cells(e.g., generated by culturing hemangioblasts) results in the releasefrom said mesenchymal stromal cells of cytokines selected from the groupcomprising transforming growth factor beta, indoleamine 2, 3dioxygenase,prostaglandin E2, hepatocyte growth factor, nitric oxide, interleukin10, interleukin 6, macrophage-colony stimulating factor, and solublehuman leukocyte antigen (HLA) G5.

In a further embodiment of the instant invention, administration of apreparation of the subject mesenchymal stromal cells (e.g., generated byculturing hemangioblasts) results in the release from said mesenchymalstromal cells of cytokines selected from the group comprisingtransforming growth factor beta, indoleamine 2, 3dioxygenase,prostaglandin E2, hepatocyte growth factor, nitric oxide, interleukin10, interleukin 6, macrophage-colony stimulating factor, soluble humanleukocyte antigen (HLA) G5, interleukin 4, 8, 11, granulocyte macrophagecolony stimulating factor, vascular endothelium growth factor,insulin-like growth factor 1, Phosphatidylinositol-glycan biosynthesisclass F protein, monocyte chemoattractant protein 1, stromal derivedfactor 1, tumor necrosis factor 1, transforming growth factor beta,basic fibroblast growth factor, angiopoietin 1 and 2, monokine inducedby interferon gamma, interferon inducible protein 10, brain derivedneurotrophic factor, interleukin 1 receptor alpha, chemokine ligand 1and 2.

Pharmaceutical Preparations of MSCs

MSCs of the instant invention may be formulated with a pharmaceuticallyacceptable carrier. For example, MSCs of the invention may beadministered alone or as a component of a pharmaceutical formulation,wherein said MSCs may be formulated for administration in any convenientway for use in medicine. One embodiment provides a pharmaceuticalpreparation of mesenchymal stromal cells comprising said mesenchymalstromal cells in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or non-aqueous solutions selectedfrom the group consisting of: dispersions, suspensions, emulsions,sterile powders optionally reconstituted into sterile injectablesolutions or dispersions just prior to use, antioxidants, buffers,bacteriostats, solutes or suspending and thickening agents.

In an embodiment of the instant invention, a pharmaceutical preparationof mesenchymal stromal cells is provided, wherein said mesenchymalstromal cells have undergone between about 5 and about 100 populationdoublings. In a further embodiment of the instant invention, apharmaceutical preparation of mesenchymal stromal cells is provided,wherein said mesenchymal stromal cells have undergone between about 10and about 80 population doublings. In a further embodiment of theinstant invention, a pharmaceutical preparation of mesenchymal stromalcells is provided, wherein said mesenchymal stromal cells have undergonebetween about 25 and about 60 population doublings. In a furtherembodiment of the instant invention, a pharmaceutical preparation ofmesenchymal stromal cells is provided, wherein said mesenchymal stromalcells have undergone less than about 10 population doublings. In a stillfurther embodiment of the instant invention, a pharmaceuticalpreparation of mesenchymal stromal cells is provided, wherein saidmesenchymal stromal cells have undergone less than about 20 populationdoublings. In a further embodiment of the instant invention, apharmaceutical preparation of mesenchymal stromal cells is provided,wherein said mesenchymal stromal cells have undergone less than about 30population doublings, wherein said mesenchymal stromal cells have notundergone replicative senescence. In a further embodiment of the instantinvention, a pharmaceutical preparation of mesenchymal stromal cells isprovided, wherein said mesenchymal stromal cells have undergone lessthan about 30 population doublings, wherein less than about 25% of saidmesenchymal stromal cells have undergone replicative senescence. In afurther embodiment of the instant invention, a pharmaceuticalpreparation of mesenchymal stromal cells is provided, wherein saidmesenchymal stromal cells have undergone less than about 30 populationdoublings, wherein less than about 10% of said mesenchymal stromal cellshave undergone replicative senescence. In a further embodiment of theinstant invention, a pharmaceutical preparation of mesenchymal stromalcells is provided, wherein said mesenchymal stromal cells have undergoneless than about 30 population doublings, wherein less than about 10% ofsaid mesenchymal stromal cells have undergone replicative senescence,and wherein said mesenchymal stromal cells express the markers selectedfrom the group comprising AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1.

Concentrations for injections of pharmaceutical preparations of MSCs maybe at any amount that is effective and, for example, substantially freeof ESCs. For example, the pharmaceutical preparations may comprise thenumbers and types of MSCs described herein. In a particular embodiment,the pharmaceutical preparations of MSCs comprise about 1×10⁶ of thesubject MSCs (e.g., generated by culturing hemangioblasts) for systemicadministration to a host in need thereof or about 1×10⁴ of said MSCs byculturing hemangioblasts for local administration to a host in needthereof.

Exemplary compositions of the present disclosure may be formulationsuitable for use in treating a human patient, such as pyrogen-free oressentially pyrogen-free, and pathogen-free. When administered, thepharmaceutical preparations for use in this disclosure may be in apyrogen-free, pathogen-free, physiologically acceptable form.

The preparation comprising MSCs used in the methods described herein maybe transplanted in a suspension, gel, colloid, slurry, or mixture. Also,at the time of injection, cryopreserved MSCs may be resuspended withcommercially available balanced salt solution to achieve the desiredosmolality and concentration for administration by injection (i.e.,bolus or intravenous).

One aspect of the invention relates to a pharmaceutical preparationsuitable for use in a mammalian patient, comprising at least 10⁶, 10⁷,10⁸ or even 10⁹ mesenchymal stromal cells and a pharmaceuticallyacceptable carrier. Another aspect of the invention relates to apharmaceutical preparation comprising at least 10⁶, 10⁷, 10⁸ or even 10⁹mesenchymal stromal cells and a pharmaceutically acceptable carrier,wherein the mesenchymal stromal cells are differentiated from ahemangioblast cell. Yet another aspect of the invention provides acryogenic cell bank comprising at least 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² oreven 10¹¹ mesenchymal stromal cells. Still another aspect of theinvention provides a purified cellular preparation free of substantiallyfree of non-human cells and/or non-human animal products, comprising atleast 10⁶, 10⁷, le or even 10⁹ mesenchymal stromal cells and less than1% of any other cell type, more preferably less than 0.1%, 0.01% or even0.001% of any other cell type. Certain preferred embodiments of theabove preparations, compositions and bank include, but are not limitedto those listed in the following paragraphs:

In certain embodiments, the mesenchymal stromal cells have replicativecapacity to undergo at least 10 population doublings in cell culturewith less than 25, 20, 15, 10 or even 5 percent of the cells undergoingcell death, senescing or differentiating into non-MSC cells (such asfibroblasts, adipocytes and/or osteocytes) by the 10^(th) doubling.

In certain embodiments, the mesenchymal stromal cells have replicativecapacity to undergo at least 15 population doublings in cell culturewith less than 25, 20, 15, 10 or even 5 percent of the cells undergoingcell death, senescing or differentiating into non-MSC cells (such asfibroblasts, adipocytes and/or osteocytes) by the 15^(th) doubling.

In certain embodiments, the mesenchymal stromal cells have replicativecapacity to undergo at least 20 population doublings in cell culturewith less than 25, 20, 15, 10 or even 5 percent of the cells undergoingcell death, senescing or differentiating into non-MSC cells (such asfibroblasts, adipocytes and/or osteocytes) by the 20^(th) doubling.

In certain embodiments, the mesenchymal stromal cells have replicativecapacity to undergo at least 5 passages in cell culture with less than25, 20, 15, 10 or even 5 percent of the cells undergoing cell death,senescing or differentiating into non-MSC cells (such as fibroblasts,adipocytes and/or osteocytes) by the 5^(th) passage.

In certain embodiments, the mesenchymal stromal cells have replicativecapacity to undergo at least 10 passages in cell culture with less than25, 20, 15, 10 or even 5 percent of the cells undergoing cell death,senescing or differentiating into non-MSC cells (such as fibroblasts,adipocytes and/or osteocytes) by the 10^(th) passage.

In certain embodiments, the mesenchymal stromal cells are differentiatedfrom a pluripotent stem cell source, such as a pluripotent stem cellthat expresses OCT-4, alkaline phosphatase, Sox2, SSEA-3, SSEA-4,TRA-1-60, and TRA-1-80 (such as, and embryonic stem cell line or inducedpluripotency stem cell line), and even more preferably from a commonpluripotent stem cell source.

In certain embodiments, the mesenchymal stromal cells areHLA-genotypically identical.

In certain embodiments, the mesenchymal stromal cells are genomicallyidentical.

In certain embodiments, at least 30%, 35%, 40%, 45% or even 50% of themesenchymal stromal cells are positive for CD10.

In certain embodiments, at least 60%, 65%, 70%, 75%, 80%, 85% or even90% of the mesenchymal stromal cells are positive for markers CD73,CD90, CD105, CD13, CD29, CD44, CD166 and CD274 and HLA-ABC.

In certain embodiments, less than 30%, 25%, 20%, 15% or even 10% of themesenchymal stromal cells are positive for markers CD31, CD34, CD45,CD133, FGFR2, CD271, Stro-1, CXCR4 and TLR3.

In certain embodiments, the mesenchymal stromal cells have replicativerates to undergo at least 10 population doublings in cell culture inless than 25, 24, 23, 22, 21 or even 20 days.

In certain embodiments, the mesenchymal stromal cells have a meanterminal restriction fragment length (TRF) that is longer than 7 kb, 7.5kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb or even12 kb.

In certain embodiments, the mesenchymal stromal cells do not undergomore than a 75%, 70%, 65%, 60%, 55%, 50%, or even 45% percent increasein cells having a forward-scattered light value, measured by flowcytometry, greater than 5,000,000 over 10, 15 or even 20 populationdoublings in culture.

In certain embodiments, the mesenchymal stromal cells, in a restingstate, express mRNA encoding Interleukin-6 at a level which is less than10%, 8%, 6%, 4% or even 2% of the IL-6 mRNA level expressed bymesenchymal stromal cells preparations, in a resting state, derived fromcord blood, bone marrow or adipose tissue.

In certain embodiments, the mesenchymal stromal cells are at least 2, 4,6, 8, 10, 20, 50 or even 100 times more potent than MSCs derived fromcord blood, bone marrow or adipose tissue.

In certain embodiments, one million of the mesenchymal stromal cells,when injected into an MOG35-55 EAE mouse model (such as C57BL/6 miceimmunized with the MOG35-55 peptide) will, on average, reduce a clinicalscore of 3.5 to less than 2.5, and even more preferably will reduce theclinical score to less 2, 1.5 or even less than 1.

In certain embodiments, the preparation is suitable for administrationto a human patient, and more preferably pyrogen free and/or free ofnon-human animal products.

In other embodiments, the preparation is suitable for administration toa non-human veterinarian mammal, such as a dog, cat or horse.

Diseases and Conditions Treatable Using MSCs Derived from CulturingHemangioblasts

MSCs have been shown to be therapeutic for a variety of diseases andconditions. In particular, MSCs migrate to injury sites, exertimmunosuppressive effects, and facilitate repair of damaged tissues. Anembodiment of the instant invention is provided, wherein apharmaceutical preparation of mesenchymal stromal cells reduces themanifestations of a pathology. An embodiment of the instant invention isprovided, wherein a pharmaceutical preparation of mesenchymal stromalcells are administered to a host suffering from a pathology. In afurther embodiment of the instant invention, a pharmaceuticalpreparation of the subject MSCs (e.g., generated by culturinghemangioblasts) reduces the manifestations of a pathology selected fromthe group comprising wound healing, graft-versus-host disease (GvHD),disease, chronic eye disease, retinal degeneration, glaucoma, uveitis,acute myocardial infarction, chronic pain, hepatitis, and nephritis. Ina further embodiment of the instant invention, a pharmaceuticalpreparation of mesenchymal stromal cells by culturing hemangioblastsreduces the manifestations of equine laminitis. As a further example,MSCs may be administered in combination with an allogeneic transplantedcell or tissue (e.g., a preparation comprising cells that have beendifferentiated from ES cells, such as retinal pigment epithelium (RPE)cells, oligodendrocyte precursors, retinal, corneal, muscle such asskeletal, smooth, or cardiac muscle or any combination thereof, orothers) thereby decreasing the likelihood of an immune reaction againstthe transplanted cell or tissue and potentially avoiding the need forother immune suppression. The subject MSCs (e.g., generated by culturinghemangioblasts) described herein may be used in similar applications. Anembodiment of a process of the instant invention, wherein theadministration of a pharmaceutical preparation of the subject MSCs(e.g., generated by culturing hemangioblasts) to a host reduces the needfor future therapy. An embodiment of a process of the instant inventionis provided, wherein the administration of a pharmaceutical preparationof the subject MSCs (e.g., generated by culturing hemangioblasts) to ahost reduces the need for future therapy, wherein said therapysuppresses immune function.

In an embodiment of the instant invention, a pharmaceutical preparationof the subject MSCs (e.g., generated by culturing hemangioblasts) isadministered to a host for the treatment of a pathology. In anembodiment of the instant invention, a pharmaceutical preparation of thesubject MSCs (e.g., generated by culturing hemangioblasts) isadministered to a host for the treatment of pathologies selected fromthe list comprising wound healing, multiple sclerosis, systemicsclerosis, hematological malignancies, myocardial infarction, tissue andorgan transplantation, tissue and organ rejection, chronic allograftnephropathy, cirrhosis, liver failure, heart failure, GvHD, tibialfracture, bone factures, left ventricular dysfunction, leukemia,myelodysplastic syndrome, Crohn's disease, Type I or Type II diabetes,obesity, metabolic diseases and disorders (such as lysosomal storagediseases including Tay-Sachs disease, Gaucher disease, Pompe disease,Hurler syndrome and metachromatic leukodystrophy), fatty liver diseasemellitus, chronic obstructive pulmonary disease, pulmonary hypertension,chronic pain, osteogenesis imperfecta, homozygous familialhypocholesterolemia, hypocholesterolemia, treatment followingmeniscectomy, adult periodontitis, periodontitis, vasculogenesis inpatients with severe myocardial ischemia, spinal cord injury,osteodysplasia, critical limb ischemia associated with diabetes,obesity, metabolic diseases and disorders (such as lysosomal storagediseases including Tay-Sachs disease, Gaucher disease, Pompe disease,Hurler syndrome and metachromatic leukodystrophy), fatty liver diseasemellitus, diabetic foot disease, primary Sjogren's syndrome,osteoarthritis, cartilage defects (e.g., articular cartilage defects),laminitis, multisystem atrophy, amyotropic lateral sclerosis, cardiacsurgery, refractory systemic lupus erythematosis, living kidneyallografts, nonmalignant red blood cell disorders, thermal burn,radiation burn, Parkinson's disease, microfractures (e.g., in patientswith knee articular cartilage injury of defects), epidermolysis bullosa,severe coronary ischemia, idiopathic dilated cardiomyopathy,osteonecrosis femoral head, lupus nephritis, bone void defects, ischemiccerebral stroke, after stroke, acute radiation syndrome, pulmonarydisease, arthritis, and bone regeneration.

In a further embodiment of the instant invention, a pharmaceuticalpreparation of the subject MSCs (e.g., generated by culturinghemangioblasts) is administered to a host for the treatment ofautoimmune pathologies selected from the list comprising Acutenecrotizing hemorrhagic leukoencephalitis, Addison's disease,Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome(APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmunedysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia,Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED),Autoimmune myocarditis, Autoimmune pancreatitis, Autoimmune retinopathy,Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease,Autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease,Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease,Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronicinflammatory demyelinating polyneuropathy (CIDP), Chronic recurrentmultifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricialpemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome,Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis,CREST disease, Essential mixed cryoglobulinemia, Demyelinatingneuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease(neuromyelitis optica), Discoid lupus, Dressler's syndrome,Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis,Erythema nodosum, Experimental allergic encephalomyelitis, Evanssyndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Glomerulonephritis, Goodpasture's syndrome,Granulomatosis with Polyangiitis (GPA) see Wegener's, Graves' disease,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpesgestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura(ITP), IgA nephropathy, IgG4-related sclerosing disease,Immunoregulatory lipoproteins, Inclusion body myositis,Insulin-dependent diabetes, obesity, metabolic diseases and disorders(such as lysosomal storage diseases including Tay-Sachs disease, Gaucherdisease, Pompe disease, Hurler syndrome and metachromaticleukodystrophy), fatty liver disease (type1), interstitial cystitis,Juvenile arthritis, Juvenile diabetes, obesity, metabolic diseases anddisorders (such as lysosomal storage diseases including Tay-Sachsdisease, Gaucher disease, Pompe disease, Hurler syndrome andmetachromatic leukodystrophy), fatty liver disease, Kawasaki syndrome,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planes,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopicpolyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis,Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia,Ocular cicatricial pemphigoid, Neuritis (including optic neuritis),Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis (peripheraluveitis), Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritisnodosa, Type I, II, & III autoimmune polyglandular syndromes,Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome,Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliarycirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriaticarthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure redcell aplasia, Raynauds phenomenon, Reflex sympathetic dystrophy,Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome,Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis,Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren'ssyndrome, Sperm & testicular autoimmunity, Stiff person syndrome,Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympatheticophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cellarteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome,Transverse myelitis, Ulcerative colitis, Undifferentiated connectivetissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis,Vitiligo, and Wegener's granulomatosis (now termed Granulomatosis withPolyangiitis (GPA).

Treatment Regimens Using MSCs Derived from Culturing Hemangioblasts

The MSCs and pharmaceutical preparations comprising MSCs describedherein may be used for cell-based treatments. In particular, the instantinvention provides methods for treating or preventing the diseases andconditions described herein comprising administering an effective amountof a pharmaceutical preparation comprising MSCs, wherein the MSCs arederived from culturing hemangioblasts.

The MSCs of the instant invention may be administered using modalitiesknown in the art including, but not limited to, injection viaintravenous, intramyocardial, tranendocardial, intravitreal, orintramuscular routes or local implantation dependent on the particularpathology being treated.

The mesenchymal stromal cells of the instant invention may beadministered via local implantation, wherein a delivery device isutilized. Delivery devices of the instant invention are biocompatibleand biodegradable. A delivery device of the instant invention can bemanufactured using materials selected from the group comprisingbiocompatible fibers, biocompatible yarns, biocompatible foams,aliphatic polyesters, poly(amino acids), copoly(ether-esters),polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups, poly(anhydrides),polyphosphazenes, biopolymers; homopolymers and copolymers of lactide,glycolide, epsilon-caprolactone, para-dioxanone, trimethylene carbonate;homopolymers and copolymers of lactide, glycolide, epsilon-caprolactone,para-dioxanone, trimethylene carbonate, fibrillar collagen,non-fibrillar collagen, collagens not treated with pepsin, collagenscombined with other polymers, growth factors, extracellular matrixproteins, biologically relevant peptide fragments, hepatocyte growthfactor, platelet-derived growth factors, platelet rich plasma, insulingrowth factor, growth differentiation factor, vascular endothelialcell-derived growth factor, nicotinamide, glucagon like peptides,tenascin-C, laminin, anti-rejection agents, analgesics, anti-oxidants,anti-apoptotic agents anti-inflammatory agents and cytostatic agents.

The particular treatment regimen, route of administration, and adjuvanttherapy may be tailored based on the particular pathology, the severityof the pathology, and the patient's overall health. Administration ofthe pharmaceutical preparations comprising MSCs may be effective toreduce the severity of the manifestations of a pathology or and/or toprevent further degeneration of the manifestation of a pathology.

A treatment modality of the present invention may comprise theadministration of a single dose of MSCs. Alternatively, treatmentmodalities described herein may comprise a course of therapy where MSCsare administered multiple times over some period of time. Exemplarycourses of treatment may comprise weekly, biweekly, monthly, quarterly,biannually, or yearly treatments. Alternatively, treatment may proceedin phases whereby multiple doses are required initially (e.g., dailydoses for the first week), and subsequently fewer and less frequentdoses are needed.

In one embodiment, the pharmaceutical preparation of mesenchymal stromalcells obtained by culturing hemangioblasts is administered to a patientone or more times periodically throughout the life of a patient. In afurther embodiment of the instant invention, a pharmaceuticalpreparation of the subject MSCs (e.g., generated by culturinghemangioblasts) is administered once per year, once every 6-12 months,once every 3-6 months, once every 1-3 months, or once every 1-4 weeks.Alternatively, more frequent administration may be desirable for certainconditions or disorders. In an embodiment of the instant invention, apharmaceutical preparation of the subject MSCs (e.g., generated byculturing hemangioblasts) is administered via a device once, more thanonce, periodically throughout the lifetime of the patient, or asnecessary for the particular patient and patient's pathology beingtreated. Similarly contemplated is a therapeutic regimen that changesover time. For example, more frequent treatment may be needed at theoutset (e.g., daily or weekly treatment). Over time, as the patient'scondition improves, less frequent treatment or even no further treatmentmay be needed.

In accordance with the present invention, the diseases or conditions canbe treated or prevented by intravenous administration of the mesenchymalstem cells described herein. In some embodiments, about 20 million,about 40 million, about 60 million, about 80 million, about 100 million,about 120 million, about 140 million, about 160 million, about 180million, about 200 million, about 220 million, about 240 million, about260 million, about 280 million, about 300 million, about 320 million,about 340 million, about 360 million, about 380 million, about 400million, about 420 million, about 440 million, about 460 million, about480 million, about 500 million, about 520 million, about 540 million,about 560 million, about 580 million, about 600 million, about 620million, about 640 million, about 660 million, about 680 million, about700 million, about 720 million, about 740 million, about 760 million,about 780 million, about 800 million, about 820 million, about 840million, about 860 million, about 880 million, about 900 million, about920 million, about 940 million, about 960 million, or about 980 millioncells are injected intravenously. In some embodiments, about 1 billion,about 2 billion, about 3 billion, about 4 billion or about 5 billioncells or more are injected intravenously. In some embodiments, thenumber of cells ranges from between about 20 million to about 4 billioncells, between about 40 million to about 1 billion cells, between about60 million to about 750 million cells, between about 80 million to about400 million cells, between about 100 million to about 350 million cells,and between about 175 million to about 250 million cells.

The methods described herein may further comprise the step of monitoringthe efficacy of treatment or prevention using methods known in the art.

Kits

The present invention provides for kits comprising any of thecompositions described herein. A preparation of mesenchymal stromalcells may be contained in a delivery device manufactured according tomethods known by one of ordinary skill in the art, and include methodsin US Patent Application Publication 2002/0103542, European PatentApplication EP 1 454 641, or preserved according to methods known by oneof ordinary skill in the art, and include methods in U.S. Pat. No.8,198,085, PCT Application WO2004/098285, and US Patent ApplicationPublication 2012/0077181. In an embodiment of the instant invention, akit comprising a preparation of about at least 8×10⁷, 8.5×10⁷, 9×10⁷,9.5×10⁷, 1×10⁸, 1.25×10⁸, or 1.25×10⁸ MSCs derived from culturinghemangioblasts. In another embodiment, a kit comprising a preparation ofabout 8×10⁷, 8.5×10⁷, 9×10⁷, 9.5×10⁷, 1×10⁸, 1.25×10⁸, or 1.25×10⁸ thesubject MSCs (e.g., generated by culturing hemangioblasts) is provided,wherein said preparation is pharmaceutical preparation. In a stillfurther embodiment of the instant invention, a kit comprising apharmaceutical preparation of about 8×10⁷, 8.5×10⁷, 9×10⁷, 9.5×10⁷,1×10⁸, 1.25×10⁸, or 1.25×10⁸ the subject MSCs (e.g., generated byculturing hemangioblasts) is provided, wherein said pharmaceuticalpreparation is preserved. In a still further embodiment of the instantinvention, a kit comprising a pharmaceutical preparation of about 8×10⁷,8.5×10⁷, 9×10⁷, 9.5×10⁷, 1×10⁸, 1.25×10⁸, or 1.25×10⁸ the subject MSCs(e.g., generated by culturing hemangioblasts) is provided, wherein saidpharmaceutical preparation is contained in a cell delivery vehicle.

Additionally, the kits may comprise cryopreserved MSCs or preparationsof cryopreserved MSCs, frozen MSCs or preparations of frozen MSCs,thawed frozen MSCs or preparations of thawed frozen MSCs.

Combinations of Various Embodiments and Concepts

It will be understood that the embodiments and concepts described hereinmay be used in combination. For example, the instant invention providesfor a method of generating MSCs comprising generating hemangioblastsfrom ESCs, culturing the hemangioblasts for at least four days,harvesting the hemangioblasts, re-plating the hemangioblasts on aMatrigel-coated plate, and culturing the hemangioblasts as describedherein for at least fourteen days, wherein the method generates at least85 million MSCs that are substantially free of ESCs.

EXAMPLES

The following examples are not intended to limit the invention in anyway.

Example 1—Generating MSCs from Hemangioblasts

Hemangioblasts were generated from the clinical grade, single-blastomerederived ESC line, MA09 [16], as follows:

First, early-stage clusters of cells were generated from MA09 ESCcultured in serum-free medium supplemented with a combination ofmorphogens and early hematopoietic cytokines, specifically bonemorphogenetic protein-4 (BMP-4), vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF),thrombopoietin (Tpo) and fms-related tyrosine kinase 3 ligand (FL). Morespecifically, ESCs from one well of a 6-well tissue-culture treatedplate were plated in one well of a six well ultra low adherence place(Corning) in 3 ml Stemline II medium (Sigma) supplemented with 50 ng/mlof VEGF and 50 ng/ml of BMP-4 (R & D) and incubated at 37° C. with 5%CO2. Clusters of cells were formed within the first 24 hr. After 40-48hours, half of the medium (1.5 ml) was replaced with fresh Stemline IImedium supplemented with 50 ng/ml of VEGF, 50 ng/ml of BMP-4, and20-22.5 ng/ml bFGF, and incubation continued for an additional 40-48hours (i.e., 3.5-4 days total).

Clusters of cells were dissociated and plated as single cells inserum-free semisolid blast-colony growth medium (BGM). Specifically,clusters of cells were dissociated by 0.05% trypsin-0.53 mM EDTA(Invitrogen) for 2-5 min. The cell suspension was pipetted up and downand then DMEM+10% FCS was added to inactivate the trypsin. Cells werethen passed through a 40 μm strainer to obtain a single cell suspension.Cells were then counted and resuspended in Stemline II medium at1-1.5×10⁶ cells/ml.

The single cell suspension (0.3 ml, 3 to 4.5×10⁵ cells) was mixed with2.7 ml of hemangioblast (HB) Growth Medium (H4536 based medium recipe:base medium methycellulose product H4536 (StemCell Technologies) pluspenicillin/streptomycin (pen/strp), Excyte growth supplement(Millipore), and the cytokines, Flt3-ligand (FL) at 50 ng/ml, vascularendothelial growth factor (VEGF) at 50 ng/ml, thrombopoietin (TPO) at 50ng/ml, and basic fibroblast growth factor (bFGF) at 20 ng/ml) with abrief vortex, and let stand for 5 min. The cell mixture was thentransferred to one well of a six-well ultra low adherence plate by usinga syringe (3 ml) attached with an 1 SG needle, and incubated at 37° C.with 5% CO2.

Some of the cells developed into grape-like blast colonies (BCs).Specifically, BCs were visible at 3 days (typically contained less than10 cells at the beginning of day 3), and after 4-6 days, grape-likehES-BCs were easily identified under microscopy (containing greater than100 cells per BC). The number of BCs present in the culture graduallyincreased over the course of several days. After 6-7 days, BCs could bepicked up using a mouth-glass capillary.

Hemangioblasts can be harvested between days 7-12 of culture andreplated onto Matrigel-coated tissue culture plates in a MEM+20% FCS.Flow cytometry analysis shows that expression levels of 5 cell surfacemarkers typically found on MSCs are relatively low in the startinghemangioblast population. (FIG. 2, left panel, average of 4 experiments+/−standard deviation). However, after three weeks of culture in MSCgrowth conditions, a homogenous adherent cell population arises thatstains >90% positive for these 5 characteristic MSC markers (FIG. 2,right panel—22-23 days, average of 4 experiments +/−standard deviation).Upon MSC culture conditions, the amount of time it takes fordifferentiating cells to acquire MSC surface markers may vary dependingon the specific ESC line used, the day of hemangioblast harvest, and thenumber of hemangioblasts plated onto Matrigel. In some experiments,markers arise in 90% of the cells by 7-14 days, whereas in otherexperiments, it may take 22-24 days for this many cells to acquire theseMSC markers.

Relating to the above experiments, FIG. 1 shows the generation ofFM-MA09-MSC from pluripotent cells, and a microscopic view of generatingmesenchymal stromal cells from ESCs via hemangioblasts. In addition,FIG. 2 contains a phenotype of FM-MA09-MSC obtained from pluripotentcell-derived hemangioblasts produced as above-described. This figureshows the percentage of cells positive for MSC surface markers in theinitial hemangioblast population (left side of graph, day 7-11hemangioblast) and after culturing hemangioblasts on Matrigel coatedplates (right side of graph) and a microscopic view of the mesenchymalstromal cells derived from the hemangioblasts (right panel photograph).Also, relating to the above experiments FIG. 17 depicts the process ofFM-MA09-MSC generation; and the effects of Matrigel, i.e., that removingcells from Matrigel at an early passage (ie, p²) may temporarily slowMSC growth as compared to those maintained on Matrigel until p6.

FIG. 18 further shows that the obtained BM-MSCs and FM-MA09-MSCs undergochondrogenesis.

Example 2—Comparison of Differentiation of ESCs and MSC-DerivedHemangioblasts

This example describes comparison of the differentiation of ESCs intoMSCs by two methods: either direct differentiation (in which ESCs weredirectly plated on gelatin or Matrigel) or the hemangioblast method (inwhich ESCs were first differentiated into hemangioblasts and then platedon Matrigel, as described in Example 1). Direct differentiation ongelatin gave rise to MSC-like cells, but the cells lacked CD105expression, suggesting incomplete adoption of MSC fate (FIG. 3, leftpanel). When ESCs were plated directly on Matrigel, the resulting cellsdid express CD105 as expected for MSCs (FIG. 3, middle panel). However,compared to MSCs produced by the hemangioblast method, the directlydifferentiated MSCs cells grew in clumps, were more difficult todisperse when splitting, and did not generate nearly as many MSCs whenstarting from equivalent numbers of ESCs (FIG. 4).

MSCs differentiated directly from ESCs also took longer to acquirecharacteristic MSC cell surface markers (FIG. 5). Once MSCs wereobtained, extended immunophenotyping shows that MSCs from both methodsare positive for other markers typically found on MSCs, such as HLA-ABC,while negative for hematopoiesis-associated markers such as CD34 andCD45 (FIG. 6). These results suggest that use of ahemangioblast-intermediate stage permits robust production ofhomogeneous MSCs from ESCs. Given these findings, additional studies onMSCs will be conducted with hemangioblast-derived MSCs.

In addition, experiments the results of which are contained in FIGS.3-6, 13, 15, 16, 19, and 21-27 (described supra) compare differentproperties of ESC-MSCs or BM-MSCs versus hemangioblast-derived MSCs andreveal that these cells exhibit significant differences which may impacttherapeutic efficacy of these cells and compositions derived therefrom.Particularly, FIG. 3 shows the percentage of cells positive for MSCsurface markers after culturing human embryonic stem cells (ESC) ongelatin coated plates (left panel), ESC on Matrigel coated plates(middle panel), and hemangioblasts on Matrigel coated plates (rightpanel). Additionally, FIG. 4 shows the MSC yield from pluripotent cells,FIG. 5 illustrates the acquisition of mesenchymal stromal cell markers,and FIG. 6 shows phenotypes of mesenchymal stromal cells derived fromdifferent culture methods, including expression of MSC markers and lackof expression of hematopoiesis and endothelial markers. Further,FM-MA09-MSG's were assayed to detect notable differences (relative toBM-MSCs) in potency and inhibitory effects (FIG. 13), stimulation ofTreg expansion (FIG. 15), proliferative capacity (FIG. 16), PG E2secretion (FIG. 19), Stro-1 and CD10 expression (FIGS. 21-22),maintenance of size during passaging (FIG. 23), CD10 and CD24 expression(FIG. 24), Aire-1 and IL-11 expression (FIG. 25), Ang-1 and CXCL1expression (FIG. 26), and and IL6 and VEGF expression (FIG. 27).

Example 3—MSCs Derived from Hemangioblasts Differentiate into Other CellTypes

MSCs, by definition, should be able to give rise to adipocytes,osteocytes, and chondrocytes. Using standard methods, FIG. 7 shows theability of hemangioblast-derived MSCs to differentiate into adipocytesand osteocytes, while FIG. 8 shows their potential to differentiatetowards chondrocytes via the expression of chondrocyte-specific genesand FIG. 18 shows their potential to differentiate towards chondrocytesvia safranin 0 staining of pellet mass cultures.

MSCs derived from hemangioblasts are expected to differentiate intoadipocytes, osteocytes, and chondrocytes. These differentiation pathwaysmay be examined using methods previously reported in the art. SeeKarlsson et al, Stem Cell Research 3: 39-50 (2009) (for differentiationof the hemangioblast-derived and direct ESC-derived MSCs into adipocytesand osteocytes). Particularly, FM-MA09-MSC display differentiationcapabilities including the ability to differentiate into adipocytes andosteocytes (FIG. 7). For chondrocyte differentiation, methods have beenadapted from Gong et al, J. Cell. Physiol. 224: 664-671 (2010) to studythis process and continue to examine the acquisition of chondrocytespecific genes, (e.g., Aggrecan and Collagen IIa) as well asglycosaminoglycan deposition through safranin O, alcian blue, and/ortoluene blue staining. Particularly, chondrogenic differentiation ofMA09 ESC hemangioblast-derived mesenchymal stromal cells (FM-MA09-MSC)was detected by mRNA expression of Aggrecan (chondroitin proteoglycansulfate 1) and Collagen IIa (FIG. 8). It has been reported in theliterature that none of these three cell types, adipocytes, osteocytes,or chondrocytes derived from MSCs will express the immunostimulatory HLADR molecule (Le Blanc 2003, Gotherstrom 2004, Liu 2006). Immunostainingand/or flow cytometry will be performed on these fully differentiatedMSC cell types to confirm these reported observations. This is importantto confirm so that differentiation of MSCs in an in vivo environmentwill not induce an immune response from the host recipient. Of thesethree cell types, chondrogenic differentiation may be of particularinterest due to its potential to be used in cartilage replacementtherapies for sports injuries, aging joint pain, osteoarthritis, etc.For such therapies, MSCs may not need to be fully differentiated intochondrocytes in order to be used therapeutically.

Example 4—Confirmation that MSCs Derived from Hemangioblasts areSubstantially Free of ESCs

MSCs should also be devoid of the ESC propensity to form teratomas. MSCswere confirmed to contain normal karyotypes (data not shown) by passage12 (˜50 days in culture). To confirm that the blast-derived MSCs do notcontain trace amounts of ESCs, teratoma formation assays were performedin NOD/SCID mice. 5×10⁶ MSCs are injected subcutaneously into the leftthigh muscle of 3 mice. CT2 ECs were used as positive controls and themice will be monitored over the course of 6 weeks to compare teratomaformation in MSC versus ESCC-injected mice. No teratomas formed in themice injected with MSCs.

Example 5—Reduction of EAE Scores by MSCs Derived from Hemangioblasts

A pilot study to treat experimental autoimmune encephalomyelitis (EAE)on 6-8 weeks old C57BL/6 mice with the hemangioblast-derived ESC-MSCswas conducted. EAE was induced by s.c. injection into the flanks of themice on day 0 with 100 μL of an emulsion of 50 μg of myelinoligodentricyte glycoprotein MOG(35-55) peptide and 250 μg of M.tuberculosis in adjuvant oil (complete Freund's adjuvant, CFA), the micewere also i.p. injected with 500 ng of pertussis toxin. Six days laterthe mice were i.p. injected with either one million ESC-MSCs in PBS(n=3) or the vehicle as a control (n=4). The clinical scores of theanimals were recorded for 29 days post the immunization. A remarkablereduction of the disease scores was observed (data not shown).

Example 6—Confirmation of the Efficacy of Hemangioblast-derived ESC-MSCsin EAE Treatment and Use of Additional Animal Models of Disease

A. Test ESC-MSCs on EAE Models in Mice to Confirm their Anti-EAE Effect.

To confirm the results obtained in Example 5, additional tests areconducted with increased animal numbers, varying cell doses, differentadministration protocols, and more controls. Clinical score andmortality rate are recorded. The degree of lymphocyte infiltration inthe brain and spinal cord of mice will also be assessed. MSC anti-EAEeffects are generally thought to involve immunosuppressive activitiessuch as the suppression of Th17 cells and would be expected to reducethe degree of lymphocyte infiltration in the CNS.

B. Compare ESC-MSCs with Mouse Bone Marrow (BM)-MSCs, Human BM-MSCs andHuman Umbilical Cord Derived (UCB)-MSCs.

Mouse BM-MSCs were the first to be used for EAE treatment and have beenthoroughly studied [1]. ESC-MSCs (given their xenogenic nature) may bedirectly compared with murine BM-MSCs for anti-EAE efficacy. HumanUCB-MSCs have been shown to also possess immunosuppressive activity[19]. The anti-EAE activity of human UCB-MSCs and human BM-MSCs may alsobe compared with that of ESC-MSCs in the EAE mouse models. The age orpassage number of these various cell types may influence their anti-EAEbehavior, thus we will also evaluate the consequences of age on theefficacy of MSCs in the EAE mouse model system.

C. Optimize the Administration Dose, Route, and Timing of ESC-MSCs.

Injection of the ESC-MSCs can reduce the scores of EAE as recordedwithin 29 days after immunization. To study long-term prevention andcure of disease, ESC-MSCs may be administered at various doses, routes,and times.

MSCs have been generated from H1gfp ESCs and confirmed that they stillexpress GFP in the MSC state. EAE mice can be injected with these GFP+ESC-MSCs and their distribution can be tracked in vivo by using aXenogen In Vivo Imaging System. Through these approaches, variousadministration doses, routes, and timing of ESC-MSCs will be analyzedand provide information as to the mechanism of action for MSCs anti-EAEactivity (le, paracrine or endocrine effects), longevity of the MSCswithin the mice and MSC biodistribution and routes ofelimination/clearance.

Anti-EAE effects may be reflected by one or more of reduced clinicalscores, increased survival, and/or attenuated lymphocyte infiltrationand demyelination of the CNS. Different ESC lines may have differentintrinsic abilities to generate MSCs. Therefore, multiple ESC lines maybe used in this study and acquisition of MSC markers can be monitoredover time and compared for each ESC line. To further reduce variationsbetween experiments with ESC-MSCs, large stocks of frozen ESC-MSCs canbe made in aliquots and each stock of aliquots can be used in multipleexperiments.

D. Confirm Efficacy of Hemangioblast-Derived MSCs in Other DiseaseModels.

As mentioned above, MSCs may also have therapeutic activity againstother types of autoimmune disorders such as Crohn's disease, ulcerativecolitis, and the eye-disorder, uveitis. Animal models for these diseasesexist and are well known in the art (see, e.g., Pizarro et al 2003,Duijvestein et al 2011, Liang et al 2011, Copland. et al 2008). In vivostudies may be expanded to include an assessment of MSC therapeuticutility in one or more of these animal model systems. Such models mayallow us to examine the cytokine secretion profile of human MSCs byisolating and screening the serum of injected animals for humancytokines. Particularly, the uveitis model may be useful as a localintravitreal injection may allow us to study the effects of MSCs in anon-systemic environment.

MSCs may also have great therapeutic utility in treating osteoarthritisconditions, including those that involve loss of articular cartilage andinflammation of the affected joints (Noth et al, 2008). Models forexamining osteoarthritis, cartilage loss and joint inflammation are wellknown in the art (see, e.g., Mobasheri et al 2009). In some of thesestudies, human BM-MSCs are encapsulated in semi-solid scaffolds ormicrospheres and transplanted into an affected joint in human subjectsto determine if the MSCs have a local, non-systemic therapeutic effectin terms of reduced inflammation and/or restoration of cartilage(Wakitani et al 2002). Such methods will assist in determining thetherapeutic utility of our ESC hemangioblast-derived MSCs for treatingdegenerative joint conditions.

The life span of injected MSCs is very short [8], which indicates thatlong-term survival of the transplanted cells is not required. Thus,mitotically-inactivated ESC-MSCs (e.g., irradiated or treated withmitomycin C) may also be tested for an anti-EAE effect or otheranti-disease effect in the animal models mentioned above. If so, liveESC-MSCs may not be needed, thus further decreasing the biosafetyconcern from potential residual ESC contamination in the transplantedESC-MSCs.

E. Results

MSCs from different donor derive sources (mouse BM-MSCs, human BM-MSCsand human UCB-MSCs) are expected to harbor anti-EAE effects. However,their effects may vary between experiments as the MSCs are fromdonor-limited sources. In contrast, the ESC-MSCs of the presentdisclosure may have more consistent effects. Because many cell surfacemarkers are used to characterize MSCs and not every MSC expresses allthe markers, a subset of markers, e.g., CD734- and CD45− may be used inorder to compare efficacy of MSCs from different sources.

ESC-MSCs are expected to have therapeutic utility in animal models ofCrohn's Disease, ulcerative colitis, and uveitis as these containautoimmune components and inflammatory reactions.

Mitotically inactivated MSCs (e.g. irradiated or mitomycin C inactivatedMSCs and/or ESC-MSCs) may retain, at least partially, theimmunosuppressive function since they still secret cytokines and expresscell surface markers that are related to the function [29]. Their effectmay, however, be decreased due to their shortened life span in vivo. Ifso, the dose of irradiated or other mitotically inactived cells andadministration frequency may be increased to enhance theimmunosuppressive function.

A second pilot study to treat EAE was conducted. Eight to ten week oldC57BL/6 mice were immunized with the MOG35-55 peptide in completeFreund's adjuvant via subQ injection. Thus was done in conjunction withIntraperitoneal injection of pertussis toxin. Six days later, 1 millionlive (or 2 million irradiated) hemangioblast-derived pluripotentcell-mesenchymal stromal cells were injected intraperitoneally permouse. Disease severity was scored on a scale of 0-5 by monitoring mouselimb/body motion, as previously published. Results demonstrated asignificant reduction in clinical score as compared to vehicle controlwith hemangioblast-derived pluripotent cell-mesenchymal stromal cells atpassage 4 and irradiated hemangioblast-derived pluripotentcell-mesenchymal stromal cells (data not shown). Scoring for both pilotstudies was performed according to the following protocol: a score of 1indicates limp tail, 2 indicates partial hind leg paralysis, 3 iscomplete hind leg paralysis, 4 is complete hind and partial front legparalysis, 5 is moribund.

In addition, the efficacy of MSCs according to the invention andproducts derivable therefrom for use in different therapies may beconfirmed in other animal models, e.g., other transplantation orautoimmune models depending on the contemplated therapeutic indication.

Example 7—Investigation of Functional Components of ESC-MSCs

MSCs may be defined as plastic adherent cells that express the followingcell surface markers: CD105, CD73, CD29, CD90, CD166, CD44, CD13, andHLA-class I (ABC) while at the same time being negative for CD34, CD45,CD14, CD19, CD11 b, CD79a and CD31 when cultured in an uninduced state(e.g., culture in regular αMEM+20% FCS with no cytokines). Under theseconditions, they must express intracellular HLA-G and be negative forCD40 and HLA class II (DR). Functionally, such cells must also be ableto differentiate into adipocytes, osteocytes, and chondrocytes asassessed by standard in vitro culture assays. After 7 days stimulationwith interferon gamma (IFNγ), MSCs should express HLA-G on their cellsurface as well as CD40 and HLA-class II (DR) on their cell surface.Despite these requirements, MSCs derived from any source may containsome heterogeneity, and due to the pluripotency of ESCs, it is possiblethat MSC cultures derived from ESCs may contain cells of any lineagefrom the three germ layers. While the culture system described hereinindicated that >90% of cells routinely display the above mentionedimmunophenotype and functional characteristics, small subpopulation(s)of cells within the MSC culture may exist that lack expression of one ormore of the MSC cell surface markers or express one or more of themarkers that should be absent. The extent of such subpopulations withinour MSC cultures will be determined by examining the degree ofcontaminating heterogeneity. Multicolor flow cytometry (8+ colorssimultaneously) can be performed on a BD LSR II flow cytometer in orderto determine the overlap between the above mentioned markers. This mayalso help pinpoint the exact cell surface marker profile that isrequired for the greatest immunosuppressive activity.

A. Characterize the Differentiation Stage, Subpopulations, andActivation Status of ESC-MSCs in Relevance to their ImmunosuppressiveEffects.

There is a large time window (e.g., at least from day 14 to 28 in theMSC differentiation medium) to harvest ESC-MSCs (see, e.g., FIG. 1).Several studies have indicated that MSCs tend to lose theirimmunosuppressive functions and may senesce as they are continuallypassaged and age during long culture periods. As such, the cells may beharvested at different time points in order to determine if a specificnumber of days in MSC Medium affords greater immunosuppressive activity.Indeed, MSCs collected at an early time point (e.g., 14 days in MSCculture conditions) may contain precursor cells that have not yet fullyacquired all of the characteristic MSC cell surface markers, but thatharbor highly potent immunosuppressive effects. To define potentiallyuseful MSC precursor populations, the expression of a wide range of cellsurface markers are being tracked throughout the MSC differentiationprocess, from day 7 through day 28. It has been observed that at least50% of the culture will acquire the cell surface marker CD309 (othernames include V EGFR2, KDR) within 14 days of MSC culture conditions.CD309 is largely absent from the starting hemangioblast population (FIG.9, first time point, MA09 hemangioblasts harvested at day 7 and day 8),but rises within the first two weeks of MSC culture conditions and thendeclines again back to less than 5% of the cells by day 28 (FIG. 9,second, third, and fourth time points). This pattern has been found tooccur not only with MA09 hemangioblast-derived MSCs but also with thosefrom MA01, H1gfp, and H7 ESCs. In these experiments, hemangioblasts areroutinely negative (less than 5% of cells stain positive) for CD309regardless of their harvest date (day 6-14). However, the percentage ofdeveloping MSCs that acquire CD309 expression may be reduced whendeveloping from older hemangioblasts (e.g., d10 or d12 blasts). In asimilar fashion, it has been observed that the expansion properties ofhemangioblast-derived MSCs may differ depending on the harvest date ofhemangioblasts. MSCs developing from younger hemangioblasts (day 6 or 7)do not continue to expand as robustly as MSCs developing from older (day8-12) hemangioblasts. The optimal date of hemangioblast harvest may bean intermediate one (day 8-10) as they may allow adequate acquisition ofCD309 as a surrogate marker of MSC development while still maintaining arobust ability to expand through day 28 and beyond.

Except CD105, CD90 and CD73 that have proved the most typical markersfor MSCs (as noted by the International Society for Cellular Therapy asthe minimum classification of MSCs (Dominici et al., Cytotherapy 8 (4):315-317 (2006)), many other cell surface molecules not mentioned abovesuch as CD49a, CD54, CD80, CD86, CD271, VCAM, and ICAM have also beenproposed or used as MSC markers [22]. It is therefore possible thatESC-MSCs may contain subpopulations that express various combinations ofother markers during the differentiation from hemangioblasts, which maypossess varying immunosuppressive activities. Subpopulations may besorted (e.g., using FACS) based one or more markers (individually or incombination) for analysis to compare their immunosuppressive activityusing in vitro or in vivo methods.

B. Optimize Differentiation and Expansion Conditions to Obtain LargeQuantities of Functional ESC-MSCs.

Preliminary experiments have indicated that MSCs may be maintained inIMDM+10% heat-inactivated human serum. Different culture conditions aretested to determine whether substituting culture components (eg, basemedium, serum source, serum replacement products, human serum plateletlysate) may enrich the effective subpopulations described herein.Different basal medium including animal-free and a defined culture(without FBS) system to culture ESCs and prepare MSCs are evaluated.Specifically, StemPro® MSC SFM from Invitrogen and the MSCM bullet kitfrom Lonza are examined to see if a serum-free defined culture systemwould generate ESC-MSCs with desired quality and quantity. Also, variousgrowth factors such as FGFs, PDGF, and TGFI3, as well as small chemicalsthat regulate signaling pathways or cell structures, are used to enhancethe quality and quantity of ESC-MSCs.

C. Results

The ESC-MSCs express the typical markers CD73 (ecto-5′-nucleotidase[26]), CD90 and CD105. Also, FIG. 20 shows that FM-MA09-MSCs producedaccording to the invention maintain their phenotype over time (based onmarker expression detected during flow cytometry analysis of differentMSC populations over time and successive passaging).

Example 8—Mechanism of Immunosuppression by ESC-MSCs

A. Study how ESC-MSCs May Suppress Adaptive Immune Responses Mediated byT Cells.

A general response of T cells within PBMC is to proliferate when theyare induced with mitotic stimulators such as phytohemagiutinin (PHA) orphorbol myristate acetate (PMA)/ionomycin or when they encounter antigenpresenting cells (AFCs) such as dendritic cells. This is bestexemplified by the general proliferation of CD4+ and CD8+ T cells in amixed leukocyte reaction (MLR) assay. Prior studies indicate that MSCscan suppress T cell proliferation in an MLR assay.

The ability of our ESC-hemangioblast derived MSCs to inhibit T cellproliferation caused by either chemical stimulation (PMA/ionomycin,FIGS. 10a and 13a ) (PHA, FIG. 13b ) or exposure to APCs (dendriticcells, FIGS. 10b and 13c ) was examined. It was observed that MSCsdampened the proliferative response of T cells due to either chemicalstimulation or co-culture with APCs and that this suppression occurredin a dose dependent manner (FIG. 10b , graph on right) Moreover, it wasfound that mitotically inactivated MSCs (FIG. 10b ) were able tosuppress T cell proliferation to an equivalent degree as live MSCs,suggesting that mitotically inactivated MSCs may indeed be useful invivo for immunosuppression.

Various functional subsets of T cells exist and they carry out specificroles involved in proinflammatory responses, anti-inflammatoryresponses, or induction of T cell anergy. Regulatory T cells (Tregs) canbe thought of as naturally occurring immunosuppressive T cells and in anormal setting, are responsible for dampening hypersensitiveauto-reactive T cell responses. They usually represent only a smallproportion of the body's T cells but their prevalence can be influencedby various environmental factors. MSCs have been shown to induceperipheral tolerance through the induction of Treg cells [33-35].

In a short, 5 day co-culture assay, it was found that, similar to priorstudies, the hemangioblast-derived MSCs were able to increase thepercentage of CD4/CD25 double positive Tregs that are induced inresponse to IL2 stimulus (FIG. 11a , 14, 15 a). Co-culture of a mixed Tcell population from non-adherent peripheral blood mononuclear cells(PBMCs) with MSCs (at a ratio of 10 PBMCs:1 MSC) shows that Treginduction nearly doubled when MSCs were included in the IL2 inducedculture. This degree of Treg induction is similar to that observed inthe highly cited Aggarwal et al study published in Blood, 2005. Theamount of FoxP3 induced within the CD4/CD25 double positive populationwas examined to confirm that these are indeed true Tregs (FIG. 15b ).Intracellular flow cytometry, was used to study FoxP3 induction in theabsence and presence of MSCs in the IL2-induced T cell cultures. Bothnon-adherent PBMCs and purified CD4+ T cell populations may be used tostudy Treg induction in these assays. Without intent to be limited bytheory, it is believed that ES-MSC are more effective at inducing Tregsbecause they increase expression of CD25 more effectively than BM-MSCs(FIG. 15b )

Th1 and Th17 cells are thought to play important roles in MS and inother autoimmune diseases. The differentiation and function of Th1 andTh17 CD4+ T cells will be analyzed first and foremost using in vitroassays; they may also be examined in the EAE model or in other animalmodels we may employ. The effects of MSCs on Th1 induction in vitro havebegun to be examined. Culture conditions that promote Th1 specificationfrom naïve CD4+ T cells are known in the field (Aggarwal et al). Theseculture conditions (which include anti-CD3, anti-CD28, and anti-CD4antibodies together with human IL3 and IL12) have been employed toinduce Th1 cells from naïve, non-adherent PBMCs in the absence orpresence of MSCs (10 PBMCs:1 MSC). After 48 hours of co-culture,non-adherent cells were isolated, rinsed, and stimulated withPMA/ionomycin for 16 hours in a new well. After the 16 hour induction,supernatants were collected and analyzed for secretion of the Th1cytokine, IFNγ. As anticipated, it was found that the PBMCs culturedwith MSCs in the 48 hr Th1 inducing conditions did not produce as muchIFNγ as those cultured without MSCs. This indicates that MSCs cansuppress a major Th1 cell function, i.e., IFNγ secretion. (FIG. 11b )Similar studies will be performed by differentiating Th17 cells in vitroand determining the effects of MSCs on pro-inflammatory IL17 secretionusing an ELISA assay on culture supernatants.

Th2 cells are known to secrete cytokines that have anti-inflammatoryeffects, such as IL4. MSCs may be able to enhance Th2 differentiationand secretion of IL4. Similar to the experiment described above for Th1cells, Th2 inducing conditions will be used in a 48 hour culture systemto stimulate Th2 differentiation from naïve PBMC containing T cells. Theeffects of MSC co-culture on IL4 secretion will be examined using anELISA assay.

Recently, studies have suggested that CD8 T cells also play a pivotalrole in EAE models and the underlying mechanism of MS [30]. The inventorwill examine if co-culture with ESC-MSCs in vitro may affect thefunction of CD8 T cells. To do this, non-adherent PBMCs or purified CD8+T cells will be exposed to EAE-associated MBP 110-118 peptide throughthe use of APCs. This will cause an antigen-specific CD8+ T cellpopulation to emerge and such a population can be expanded usingCD3/CD28 expander beads (Invitrogen). Existence of the antigen-specificCD8+ T cells can be verified using a pentamer reagent specific for theMBP-peptide (Proimmune) in flow cytometry. Re-stimulation with MBP110-118-loaded APCs will be performed in order to induce an antigenspecific immune response, which includes both expansion of theantigen-specific CD8+ T cells and secretion of IFNγ. The response from Tcells cultured in the absence or presence of MSCs will be compared todetermine if the MSCs can suppress the induction of these cytotoxicEAE-associated antigen specific T cells. Pentamer specific flowcytometry, BrdU incorporation, and ELISA assays will be employed forthis purpose.

B. Determine if Inflammatory Factors and Inter-Cellular AdhesionMolecules (ICAMs) Contribute to the Immunosuppresive Effect of ESC-MSCs.

It has been shown that TGFbeta, PGE2, IDO, nitric oxide (NO), and ICAMsare important for the immunosuppressive function of MSCs [7]. Thesecretion of these molecules and expression of ICAMs by ESC-MSCs will beexamined using ELISA assays and flow cytometry.

It has been shown that the pro-inflammatory cytokine, IFNγ is requiredfor the activation of MSCs [23], and various agonists for Toll-likereceptors (TLRs) such as LPS and poly(I:C) can induce different subsetsof MSCs [24]. For example, it has recently been shown thatIFNγ-activated MSCs have greater therapeutic efficacy in a mouse modelof colitis than do untreated MSCs (Duijvestein et al 2011). The effectsof IFNγ on MSC properties have begun to be examined. FM-MA09-MSCs weretreated in vitro with IFNγ for up to seven days and striking changes incell surface marker expression resulted. These findings are consistentwith observations made in previous studies (Gotherstrom et al 2004,Rasmusson et al 2006, Newman et al 2009) and confirm that thehemangioblast derived ESC-MSCs function similarly to MSCs isolated fromthe body. For example, in a resting state, MSCs typically do not expressmuch (<10%) HLA G on their cell surface while they do harborintracellular stores of this special class of immunotolerant HLA marker.Upon 7 days IFNγ treatment, HLA G can be readily detected at the cellsurface of FM-MA09-MSCs (FIG. 12) and the cells may also be induced tobe secreted (not yet tested). Additionally, IFNγ treatment causes anupregulation of CD40 expression and HLA DR expression at the cellsurface (FIG. 12). These changes are proposed to enhance theirimmunosuppressive effects. For example, pretreatment of MSCs with IFNγmay enhance their ability to induce Treg populations, to suppress Th1secretion of IFNγ, or to enhance IL4 secretion from Th2 cells by usingin vitro co-culture assays described above. IFNγ may also influence theability of MSCs to inhibit general T cell proliferation in MLR assays.The effects of TNFα, LPS, and/or poly I:C on these types of MSCimmunosuppressive properties may also be tested.

C. Results

It was shown that the CD4/CD25 double positive population of Tregsinduced by MSCs also express the transcription factor, FoxP3 as it hasbeen reported that functional Tregs upregulate its expression inresponse to inducing stimuli (FIG. 15b ).

It is expected that MSCs will inhibit, to some degree thepro-inflammatory secretion of IL17 by Th17 cells and that MSCs can alsosignificantly enhance IL4 secretion by anti-inflammatory Th2 cells. Suchobservations have been made in previous studies and will assist inconfirming the true functionality of the hemangioblast-derived MSCs.

The ESC-MSCs should inhibit at least partially the antigen-inducedactivation of CD8+ T cells. The function of NK cells, macrophages, anddendritic cells after ESC-MSC co-culture may also be examined. Theeffects of ESC-MSCs on maturation, cytotoxicity, and/or specificcytokine production by these other types of immune cells will beexamined.

For example, the experiments in FIG. 11A show that FM-MA09-MSCs increasethe percentage of CD4/CD25 double positive Tregs that are induced inresponse to IL2 stimulus. Also, the experiments in FIG. 12 show that theproinflammatory cytokine IFNg stimulates changes in FM-MA09-MSC surfacemarker expression and that interferon gamma stimulates changes in MSCsurface marker expression and may enhance MSC immunosuppressive effects.

Moreover, the experiments in FIG. 14 show that FM-MA09-MSCs enhance Treginduction, and particularly that early passage MSCs had greater effectsthan late passage MSCs. Non-adherent PBMCs (different donors) werecultured with or without IL2 for 4 days in the absence or presence ofFM-MA09-MSCs. The percentage of CD4/CD25 double positive Tregs wasassessed by flow cytometry. Young (p6) or old (p16-18) FM-MA09-MSCs wereused. The black bars indicate the average of 6 experiments. MSCs as awhole had a statistically significant effect on induction of Tregs.(p=0.02).

Example 9—Hemangioblast Derived ESC-MSCs have Increased Potency andGreater Inhibitory Effects than BM-MSCs

A mixed lymphocyte reaction (MLR) assay was performed to determine ifdifferent MSC populations have different abilities to inhibit T cellproliferation. Results suggest that FM-MA09-MSCs are more potent thanBM-MSCs in their ability to inhibit T cell proliferation in response toeither mitogenic stimulus (“one-way MLR”) (see FIGS. 13a and 13b ) or toantigen-presenting cells (dendritic cells, DCs; “two-way” MLR) (see FIG.13c ).

The “one-way” MLR assay was performed as follows: Human PBMCs werepurchased from AllCells. Upon thawing a frozen vial, PBMCs were platedfor at least 1 hour or overnight in IMDM+10% heat-inactivated humanserum to selectively adhere monocytes. The non-adherent cells(containing T cells) were used as a crude source of T cell responders.FM-MA09-MSCs or BM-derived MSCs were used as inhibitors. These MSCs werewere either live or mitotically-arrested with mitomycin C. Non-adherentPBMCs and MSCs were mixed together at varying ratios and allowed toco-culture for 5 days. On day 3, the mitogens, phorbol-12-myristate13-acetate (PMA) and ionomycin or phytohemagglutinin (PHA) were added tothe cultures to induce T cell proliferation. On day 4, bromodeoxyuridine(BrdU) was added. On day 5, T cell proliferation was assessed throughflow cytometric staining with antibodies directed against CD4, CD8, andBrdU using the BrdU incorporation kit (B&D Biosystems). T cellproliferation was assessed as the % of CD4+ and/or CD8+ cells that hadincorporated BrdU into their DNA (ie, BrdU+) (shown in FIGS. 13a and 13b). In FIG. 13b , MA09-MSCs p7 show an approximately 40% decrease inT-cll activation compared to BM-MSCs.

In the “two-way” MLR, FM-MA09-MSCs or BM-derived MSCs were used asinhibitors, non-adherent peripheral blood mononuclear cells (PBMCs) wereused as a crude source of T cell responders, and monocyte-deriveddendritic cells (DCs) were used as stimulators. To derive DCs,plastic-adherent monocytes were isolated from PBMCs PBMCs were platedfor at least 1 hour or overnight in IMDM+10% heat-inactivated humanserum (10% HuSer) to selectively adhere monocytes. Non-adherent cellswere removed and the adherent cells were cultured in IMDM+10% HuSer for4 days with SCF, FL, GM-CSF, IL3, and IL4. In this variation of theassay, no mitogen is added on day 3. BrdU is simply added 16-24 hoursbefore harvesting the cells for flow cytometry as above. Both MSCs andDCs were mitotically-inactivated with Mitomycin C in this assay (shownin FIG. 13c ). In FIG. 13c , the percent BRDU incorporation of the Tcells does not substantially increase, preferably remains the same, morepreferably decreases the DC/T cell ratio in the presence of theMA09-MSCs as compared to the BM-MSCs.

Example 10—Improved Induction of Treg Expansion by Young FM-MA09-MSCsCompared to BM-MSCs and Old FM-MA09-MSCs

Co-culture experiments were performed with PBMCs and FM-MA09-MSCs todetermine if the presence of these MSCs can induce regulatory T cell(Treg) expansion within the PBMC population. Results suggest that youngFM-MA09-MSCs induced Treg expansion better than both BM-MSCs and oldFM-MA09-MSCs (see FIG. 14 and FIG. 15).

Co-cultures were established with non-adherent PBMCs and different typesof FM-MA09-MSCs (“young” ESC-derived (˜p5-6), “old” ESC-derived (˜p12 orhigher), BM-derived) at a 10:1 ratio (PBMC:MSC). Co-cultures wereincubated in IMDM+10% heat inactivated Human Serum+300 units/mlrecombinant human IL2 for 4 days. The presence of Tregs was determinedby the percentage of PBMCs that stained positive for CD4, CD25, andFoxP3 using a FoxP3 intracellular flow cytometry staining kit(Biolegend).

Example 11—ESC-MSC Derived Via Hemangioblasts have Greater ProliferativeCapacity

The growth rates of different MSC populations were monitored over timeto determine if the source of MSCs affects their proliferative capacity.Results show that FM-MA09-MSCs have greater proliferative capacity thanBM-derived MSCs. Results also suggest that culturing FM-MA09-MSCs on asubstrate (such as Matrigel) for a longer period of time (up to 6passages) may help maintain a higher growth rate than if the cells aremoved off of the substrate at an earlier passage, such as p2 (see FIG.16 and FIG. 17).

ESC-derived hemangioblasts were seeded onto Matrigel-coatedtissue-culture plastic at 50,000 cells/cm² in αMEM+20% HycloneFBS+1-glutamine+non-essential amino acids (MSC growth medium) at p0.Bone-marrow mononuclear cells were seeded onto regular tissue cultureplastic at 50,000 cells/cm² in MSC medium at p0. Cells were harvestedwith 0.05% trypsin-edta (Gibco) when they reached ˜50-60% confluence atp0 or at 70-80% confluence from p1 onwards (usually every 3-5 days).Upon harvest, cells were spun down, counted, and replated at 7000cells/cm². ESC-MSCs were removed from Matrigel and subsequently grown onregular tissue culture plastic starting at p3, unless otherwiseindicated. Cumulative population doublings over time are plotted to showthe rate of cell growth as the MSCs are maintained in culture.

Example 12—FM-MA09-MSCs Undergo Chondrogenic Differentiation

To determine the chondrogenic potential of different MSC populations,FM-MA09-MSCs or BM-MSCs were seeded as pellet mass cultures and inducedto differentiate into chondrocytes with differentiation medium (or keptin regular MSC growth media as negative controls). Results suggest thatFM-MA09-MSCs undergo chondrogenesis in a manner similar to that ofBM-MSCs. Both ESC-MSC and BM-MSC pellets reveal cartilaginous matrix(proteoglycan) deposition via Safranin 0 staining (see FIG. 18).

To form chondrogenic pellet culture, 2.5×10⁵ cells FM-MA09-MSCs werecentrifuged at 500×g for 5 min in a 15 mL conical tube. Culture mediumwas aspirated and 0.5 mL of chondrogenic culture medium, consisting ofDMEM-HG (Life Technologies, Gaithersburg, Md.) supplemented with 1 mMSodium Pyruvate (Life Technologies), 0.1 mM ascorbic acid 2-phosphate(Sigma-Aldrich, St. Louis, Mo.), 0.1 μM dexamethasone (Sigma-Aldrich),1% ITS (Collaborative Biomedical Products, Bedford, Mass.), 10 ng/mLTGF-β3 (Peprotech, Rocky Hill, N.J.), or culture medium (control) wasadded to the pellet. Pellet cultures were maintained for 21 days withmedium changes every 2-3 days. At the end of the 21 days, pellets werefixed with 4% paraformaldehyde and sent to MassHistology (Worcester,Mass.) for paraffin-embedding, sectioning, and Safranin O staining usingstandard procedures.

Example 13—Enhanced Sectretion of Prostaglandin E2 (PGE2) Under IFN-γ orTNF-α Stimulation

ESC-MSCs exert immunomodulatory effects in part through the secretion ofPGE2. Conditioned medium collected from FM-MA09-MSCs and BM-MSCs showthat BM-MSCs secrete higher levels of PGE2 in the basal state thanFM-MA09-MSCs. Experiments to determine PGE2 secretion under stimulatedconditions (various concentrations of IFN-γ and/or TNF-α) show thatFM-MA09-MSCs greatly increase their secretion of PGE2 in response tostimulation (see FIG. 19). In fact, the fold induction for PGE2secretion from a basal to stimulated state is much greater forFM-MA09-MSCs than for BM-MSCs. However, the actual raw amounts of PGE2secretion (in pg/ml) under stimulated conditions is similar forFM-MA09-MSCs and BM-MSCs.

FM-MA09-MSCs were plated at 7.5×10⁶ cells/cm₂ in 6 well plates (BDFalcon, Franklin Lakes, N.J.). Cultures were maintained in culturemedium for 24 hrs, followed by stimulation with 10, 50, 100, or 200ng/ml IFN-γ and/or 10, 25, 50 ng/mL TNF-α (Peprotech). Supernatant wascollected after 3 days of induction and stored at −20° C. FM-MA09-MSCswere harvested and counted to normalize PGE2 levels to cell number. PGE2concentration was measured with ELISA kits (R&D PGE2 Parameter orProstaglandin E2 Express EIA kit, Cayman Chemicals) and used accordingto manufacturer's protocol.

Example 14—FM-MA09-MSCs Phenotypic Evaluation

The expression of various cell surface markers was assessed on differentMSC populations to determine their individual immunophenotypes.FM-MA09-MSCs can be differentiated on various substrates. A panel ofcell surface markers were examined to determine their expression profileon MSCs that had been derived on three different matrices (Matrigel,fibronectin, or collagen I) versus their expression on BM-MSCs. Resultsshow similar patterns of expression for these markers regardless of thesubstrate used for their initial differentiation. They were over 95%positive for CD13, 29, 44, 73, 90, 105, 166, and HLA-ABC while negativefor CD31, 34, 45, HLA-DR, FGFR2, CD271 (see FIG. 20A). Stro-1 expressionvaried, between approximately 5% for FM-MA09-MSCs to approximately 30%for BM-MSCs.

MSCs slow in growth and population doubling with increasing passagenumber. The aim of this experiment was to look at surface markerexpression for a number of different MSC markers from passage 3 to 17 inFM-MA09-MSCs. Cells in all passages of FM-MA09-MSCs stained positive forCD90, CD73, CD105, HLA-ABC, CD166, CD13, and CD44. Cells were negativefor CD34, CD45, TLR3, HLA-DR, CD106, CD133, and CD271 (see FIG. 20B).

For each line/passage number, the same protocol was followed. Cells weregrown in T75 or T175 flasks, in MSC media. Cells were passaged every 3-4days. Passaging cells consisted of washing flasks with PBS, collectingcells using cell dissociation media TryPLE Express, and washing with MSCmedia. Cells were counted for viability with trypan blue and aliquotedat 50-100,000 viable cells per condition. The following antibodies wereused: CD34-Fitc, CD34-PE, CD44-Fitc, CD73-PE, CD106-PE, CD45-APC (BD);HLA-DR-APC, CD90-Fitc, HLA-ABC-Fitc, CD133-APC, CD29 (ebioscience);CD166-PE, CD105-APC, CD13-PE, CD13-APC, CD271-Fitc, CD10-Fitc,Stro-1-AF647, CD10 (Biolegend); TLR3-Fitc (Santa Cruz Biotech).Propidium Iodide was also added as a viability marker. Cells wereincubated at room temperature for 30 minutes, spun down, passed througha 40 μm cell strainer, and analyzed with na Accuri C6 Flow Cytometer.For each cell type, cells were gated on the MSC population (FSC vs.SSC), PI negative. Percent positive was determined by gating histogramplots and using the unstained cell population as a negative control.See, Wagner W, et al. Replicative Senescence of Mesenchymal Stem Cells:A Continuous and Organized Process. PLoS ONE (2008). 3(5): e2213.doi:10.1371/journal.pone.0002213; and Musina, R, et al. Comparison ofMesenchymal Stem Cells Obtained from Different Human Tissues. CellTechnologies in Biology and Medicine (2005) April. 1(2), 504-509.

Additionally, FM-MA09-MSCs have a greater level of CD10 expression andless Stro-1 expression than BM-MSCs (see FIG. 21). This expressionpattern of low Stro-1 (5-10% of cells) and mid-level CD10 (˜40% ofcells) was confirmed in 10 different lots of FM-MA09-MSCs (see FIG. 22).Flow cytometry was also used on different populations to evaluate cellsize (see FIG. 23). Results show that as the cells are maintained inculture for longer periods of time, the cell size of BM-MSCs increaseswhile FM-MA09-MSCs maintain cell size. Cell size was determined byforward vs. side scatter on flow cytometry dot plots. A quadrant gatewas used to divide the plot into 4 regions. The upper right quadrantcontains the large cells, i.e., cells in that area have large forwardscatter (cell volume) and also high side scatter (granularity).

FM-MA09-MSCs were harvested, as previously mentioned, and washed in1×DPBS (Life Technologies). 75-100×10⁵ cells were washed with flowbuffer (3% FBS; Atlas Biologicals, Fort Collins, Colo.), followed byincubation with 100 μL of flow buffer containing either primary antibodyor isotype control antibody for 45 min on ice. Cells were washed with 2mL flow buffer and incubated in 100 μL flow buffer containing secondaryantibody for 45 min on ice. Cells were washed a final time andresuspended in flow buffer containing propidium iodide and analyzed onan Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, Mich.).

Example 15—Gene Expression Analysis in FM-MA09-MSCs

The purpose of these studies was to determine the similarities anddifferences of mRNA expression between FM-MA09-MSCs and BM-MSC. In thefirst set of experiments (basal experiments), relative differences ofmRNA expression of cells from FM-MA09-MSCs and BM-MSC were compared byQuantitative Polymerase Chain Reaction (QPCR). Taqman probes (LifeTechnologies) to the various genes were used to determine relativeexpression to the endogenous control, GAPDH, using the ΔΔCt method. Froma list of 28 genes, the following genes were upregulated in the basalexperiments in FM-MA09-MSCs vs BM-MSC: AIRE, ANGPT1 (ANG-1), CXCL1,CD10, CD24, and IL11 (see FIGS. 24-26). IL6 and VEGF were downregulatedin FM-MA09-MSCs vs BM-MSC (see FIG. 27). There was no significantdifference for the following genes between the sources of MSC: ALCAM,FGF7, HGF, LGALS1, NT5E, and TNFSF1B (data not shown). The followinggenes were not detected in any of the MSC sources: ANGPT2, CD31, CD34,CD45, HLA-G, IL2RA, IL3, IL12B (data not shown). As a negative control,all MSCs were tested for expression of the hematopoietic progenitormarkers, CD34, CD41, and CD45. From these experiments, we havedetermined that FM-MA09-MSCs do express some genes at higher or lowerlevels than the equivalent BM-MSCs.

We also challenged the MSCs to an environment that mimics an immuneresponse by treating the MSC with T cells and then adding the stimulant,Phytohemagglutinin (PHA FM-MA09-MSCs were grown in the presence of Tcells (unstimulated) or T cells plus PHA (stimulated) for two daysbefore adding 2.5 μg/ml PHA for an additional 2 days prior to RNAcollection. The gene expression of ESC-MSC unstimulated and stimulatedare currently being compared to unstimulated and stimulated BM-MSC mRNAlevels.

For basal experiments: FM-MA09-MSCs and BM-MSC were cultured for 4 daysat a starting density of approximately 500,000 cells in a 10 cm dishunder previously described conditions. Additionally, a negative controlfor basal experiments was MA09 ESC derived hematopoetic progenitors.

For stimulation experiments: FM-MA09-MSCs and BM-MSC were cultured for3-4 days at a starting density of approximately 500,000 cells in a 10 cmdish under previously described conditions. MSCs were then exposed to Tcells for 2 days and then +/−exposure to 2.5 μg/ml PHA. As a control,MSCs were grown in the presence of T cells without PHA, and separately,T cells plus PHA (no MSCs) were also grown. Media was aspirated, rinsed2 times in PBS, and aspirated dry. RNA was isolated using the RNAeasykit (Qiagen) as per manufacturer's directions. The concentration andpurity of RNA was analyzed by using the Nanodrop 2000 (ThermoScientific). cDNA synthesis was performed using the SuperScript® IIIFirst-Strand Synthesis SuperMix for qRT-PCR (Life Technologies) using 1microgram RNA as the starting material. cDNA was diluted approximately30 fold for 5 microliters/well. Diluted cDNA, 1 microliter of QPCRTaqman probe (Life Technologies), and 15 microliters of SSO FastMastermix (Biorad) were mixed per well. QPCR was performed on the BioradCFX 96. Data was analyzed using CFX manager 2.1 (Biorad). Relativequantities of mRNA expression were determined using the endogenouscontrol, GAPDH, and the ΔΔCt method.

Example 16—Indoleamine 2, 3-Dioxygenase (IDO) Enzyme Activity inFM-MA09-MSCs

Indoleamine 2, 3-dioxygenase (IDO) is an enzyme involved in theconversion of tryptophan to kynurenine. IFNγ-activated MSCs produce IDOand this may be partly responsible for their ability to suppress T cellproliferation as IDO interferes with T cell metabolism. In this study,we are testing the IDO activity of BM-MSCs compared with FM-MA09-MSCs.IDO expression is being measured before and after stimulation of cellswith either IFNγ or by co-culturing with T cells. Experiments show allMSC populations greatly increase IDO activity upon stimulation withIFNgamma (see FIG. 28).

Cells were stimulated by the addition of either IFNγ (50 ng/m) to media,or by co-culture with T cells for 3 days; measurement of IDO expressionis performed using a spectrophotometric assay. After stimulation, cellswere collected, and 1-2×10⁶ cells are lysed. Lysates are collected, andmixed 1:1 with 2×IDO buffer (PBS with 40 mM ascorbate, 20 μM methyleneblue, 200 μg/ml catalase, and 800 μM L-tryptophan) and incubated for 30minutes at 37° C. The reaction was stopped by addition of 30%trichloroacetic acid, and incubated for 30 minutes at 52° C. Lysateswere spun down, and supernatants are mixed 1:1 with Ehrlich's reagent(0.8% p-dimethylaminobenzaldehyde in acetic acid, freshly prepared).After color development, absorbance was read on a spectrophotometer at492 nm. OD values were compared with a standard of kynurenine from0-1000 μM for assessing the conversion of tryptophan to kynurenine.

See, Meisel R et. al. Human bone marrow stromal cells inhibit allogeneicT-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophandegradation. Blood. (2004) Jun. 15; 103 (12): 4619-21.

See, Braun, D et. al. A two-step induction of indoleamine 2,3dioxygenase (IDO) activity during dendritic-cell maturation. Blood.(2005) Oct. 1; 106 (7): 2375-81.

Example 17—Expression Levels of Aire-1 and Prion-Protein in FM-MA09-MSCs

The expression levels of Aire-1 and Prion-Protein (Prp) were monitoredusing western blot analysis to determine if there are differences amongdifferent MSC populations (based on cell source, derivation method, orpassage number of the MSCs). Aire-1 helps induce transcription of rareperipheral tissue-restricted antigens (PTA) that are subsequentlypresented on MHC and quell the response of neighboring T cells. Aire-1may also suppress expression of early T cell activation factor-1 (ETA-1)to inhibit T cell inflammatory response. Prion protein (PrP) has beenshown to enhance the proliferation and self-renewal of various stem cellpopulations (hematopoietic, neural, etc) and its expression maycorrelate with the growth characteristics of different MSC populationsin culture. Results show age-related decline in both proteins (afterconsideration of loading control, actin for each sample). FM MA09-MSCsappear to maintain expression of both Aire-1 and PrP over time (see FIG.29).

MSCs whole cell lysates were run on 12% acrylamide SUS-PAGE gelsaccording to standard protocols. Proteins were transferred tonitrocellulose membrane and blocked with 5% milk in PBS+0.05% tween 20.Membranes were probed with antibodies directed against Aire-1 (SantaCruz Biotechnology) or Prion Protein (Abeam), followed by HRP-conjugatedsecondary antibodies. Enhanced chemiluminescent reagent was used todevelop the signal prior to analysis on a Biorad GelDoc Imaging System.

See, Parekkadan et al. Molecular Therapy 20 (1): 178-186 (2011).

See, Mohanty et al. Stem Cells 30: 1134-1143 (2012).

Example 18—FM-MA09-MSCs Secretion of Cytokines

MSCs are known to secrete a variety of cytokines and growth factors inboth the basal state and in response to various stimuli. More than 20different secreted factors were analysed using cytokine arrays. Resultsshow that there are a few key differences between FM-MA09-MSCs andBM-MSCs with respect to secreted factors in both the basal andstimulated states. BM-MSCs express higher levels of VEGF and IL6 than doFM-MA09-MSCs in both the basal and IFNγ-stimulated state (see FIGS.30-32).

Equivalent numbers of MSCs were initially plated and conditioned mediumfrom MSCs were collected 3-4 days after plating. CM was spun downbriefly to remove cellular debris and then frozen at −20 C. CM wasthawed for analysis on RayBiotech (Norcross, Ga.) custom membrane arraysor on various R&D Systems (Minneapolis, Minn.) ready-made cytokinearrays according to manufacturer's protocols.

Example 19—Human ES Cell Culture for the Differentiation of MSCs

The purpose of this experiment was to evaluate different growth mediaused for hESC culture prior to differentiation into MSCs.

Human ES cells were generally cultured on irradiated or mitomycin-Ctreated mouse embryonic fibroblasts (MEF) feeder cells in Human ES CellGrowth Medium (knockout DMEM or DMEM/F12 (1:1) base medium, 20% serumreplacement, I-glutamine, non-essential amino acids, and 10 ng/ml bFGF).Passaging is performed using 0.05% trypsin/EDTA. Alternatively, hESCswere cultured on MEF feeders in Primate Medium and passaged usingDissociation solution (both are purchased from ReproCELL). Resultsshowed that Primate Medium consistently gave “better” looking hESCcolonies (rounder, tighter colonies, less spontaneous differentiation)compared to cells grown on the Human ES Cell Growth Medium containingknockout DMEM.

Example 20

Pain Study

The effects of MSC produced from hES cells (MA09-MSCs) on orofacial painsensitivity were tested in the mouse. The mouse system used operantconditioning to study orofacial pain sensitivity to extreme temperatures(both hot and cold) as described in Nolan et al., Behav Brain Res. 2011Mar. 1; 217(2): 477-480, which is hereby incorporated by reference inits entirety. Sensitivity was further augmented by treatment with thechemotherapeutic agent, taxol, and the effect of MSC on hot and coldsensitivity was determined.

Methods:

MSCs were derived from hESC line MA09 generally as described in example1 above. In brief and starting with MA09 hESCs gown on mouse embryonicfibroblasts (MEFs), we use a 3-step approach to generate MSCs. hESCswere first differentiated into embryoid bodies (EB) for 4 days and theninto hemangioblasts for 9 days. The hemangioblasts were then transferredinto an MSC derivation medium. Within about 14 days, the attached cellsfully differentiated into MSCs cells with cell surface markers similarto those of BM-MSCs. hESC-MSC cultures were passaged every 3-4 days andreplated at 5000 cells/sq cm. At the third passage (p3), cells werefrozen down and then subsequently thawed and grown for 4 days as fourthpassage (p4) MSCs prior to use in the model. Cells in culture werefreshly harvested with 0.05% trypsin on the day of injection into SKH1hairless mice for the pain study. Orofacial pain testing was performedusing the Stoelting Orofacial Pain Assessment Device (OPAD)(www.stoeltingco.com/stoelting/3438/1465/1480/Physio/OroFacial-Pain-Assessment-Device-OPAD-NEW)as previously described (Rossi et al., Molecular Pain 2006, 2:37, whichis hereby incorporated by reference in its entirety). In brief, miceundergo a two week training period whereby, after fasting, they areplaced into a chamber containing a milk reward bottle located behind 2temperature-controlled thermode elements. Mice are conditioned to pushthrough the thermodes with their face in order to gain access to themilk reward. Milk licks per facial contact with the thermodes ismeasured in an automated fashion. After conditioning, baselinemeasurements (thermodes at 37 degrees) were taken in 2 independentsessions prior to any treatment and the licks per facial contact ratiowas averaged for each group of mice, n=10 per group. Animals were theni.p. injected with 1×10E6 MA09 hESC-derived MSCs in 250 ul PBS or 250 ulPBS alone. 1 week later, mice were injected with 2 mg/kg of thechemotherapeutic agent, taxol. Heat or cold sensitivity was measured foreach group of mice by changing the temperature in the thermode elementsto either 42° C. or 18° C., respectively and recording their lick/facialcontact ratio at these temperatures weekly for 4 weeks.

Results:

Results were computed as percentage of baseline licks/facial contactratio over time. Pain sensitivity resulted in a decrease of thelicks/facial contact ratio as compared to baseline, which is set as100%. Thus, the mice avoided pushing through the electrodes due to thesensation of pain when they came in contact with heat or cold.

hMSCs blocked induction of chemotherapy induced heat hyperalgesia.Animals were pretreated with hMSCs (1×10E6 cells, 250 μl, i.p.) 1 weekprior to injection of taxol (2 mg/kg). MSCs blocked the development ofheat sensitivity when tested at 42° C. Animals treated with vehicle(PBS, 250 μl, i.p.) developed heat hyperalgesia over the 3-week periodpost-taxol injection. There was a significant between-subjects effectwhen comparing treatment (hMSC vs Veh; RM ANOVA, F(1,15)=4.95, p=0.042).Baseline values were not significantly different between the hMSC andVeh groups (P=0.946). Thus, a statistically significant block intaxol-induced sensitivity to heat for MSC-injected animals vs controlswas observed. Taxol caused mice to feel more pain at 42° C. than theydid before taxol treatment, with the percentage of baseline beingdecreased at weeks 1, 2, and 3. In animals administered MSCs, thetaxol-induced sensitivity to heat was prevented. By week 4, the effectsof the taxol in the vehicle control group had subsided and they returnedto the same degree of heat sensitivity as they had before they weregiven the taxol (week 0 levels).

hMSCs also blocked induction of cold sensitivity. Animals werepretreated with hMSCs (1×10E6 cells, 250 μl, i.p.) 1 week prior toinjection of taxol (2 mg/kg). This treatment blocked cold sensitivitywhen tested at 18° C. as compared to animals treated with vehicle (PBS,250 μl, i.p.). There was a significant between-subjects effect whencomparing treatment (hMSC vs Veh; RM ANOVA, F(1,15)=16.71, p=0.01).Thus, a statistically significant block in cold sensitivity was observedfor MSC-injected animals vs controls. At weeks 1, 2, and 3, no drop inthe percentage of baseline due to taxol was observed. Nonetheless,general sensitivity to cold was blocked by MSCs, as indicated by thefact that the percentage of baseline in the MSC-injected mice wassignificantly higher than in the control group in all 3 weeks tested.Thus, taxol treatment was not observed to cause an appreciabledevelopment of cold sensitivity in this experiment, but nonethelessthere was a difference in overall cold sensitivity between theMSC-treated and control groups, with administration of MSC resulting indecreased cold sensitivity.

Example 21

Uveitis Study

Uveitis is an inflammatory eye disorder that affects the uvea of the eye(uvea is the middle layer of the eye and is comprised of the ciliarybody, iris, and choroid). Causes include autoimmune-related mechanisms(i.e., inflammation is due to autoimmune attack on some component withinthe uvea, sometimes observed in lupus) and infectious mechanisms (i.e.,inflammation is due to the immune system fighting an infection in theeye). In this example the effects of MSCs derived from hESC (MA09-MSCs)are evaluated using an established mouse model of uveitis (Tasso et al.,Invest Ophthalmol Vis Sci. 2012; 53:786-793, which is herebyincorporated by reference).

Methods:

FM-MA09-MSCs were generated as described in the preceding example. Anexperimental autoimmune uveitis (EAU) model of uveitis in C57BL/6 micewas established generally as previously described (Tasso et al., InvestOphthalmol Vis Sci. 2012; 53:786-793). In brief, male mice aged 7-9weeks were immunized with 500 ug of a peptide (aa1-20) derived frominterphotoreceptor retinoid-binding protein 3 (IRBP) along with 1.5 ugpertussin toxin (PTX). As indicated, mice were also i.p. injected with asingle bolus of 5×10⁶ MA09 hESC-MSCs on the same day as the immunization(day 0). On day 21, mice were subjected to funduscope imaging andclinical evaluation and then sacrificed to collect eyes for histologicalevaluation as well as spleens and serum for tissue banking and follow-up(e.g., Treg evaluation). Three groups of mice were used in theexperiment as follows: Group A contained 6 mice induced with EAU but NOTinjected with MSCs (to evaluate the degree of disease induction withoutany treatment, i.e., a disease-specific positive control). Group Bcontained 8 mice that were not induced with EAU but did receive MSCs(negative control for non-specific effects of MSCs in wt mice) and GroupC contained 7 mice that were induced with EAU and given MSCs, i.e. theexperimental group).

Results

Histological analysis was performed for the 3 groups of mice. Uveitisinduced in this model was rather mild, as noted by the histology imagesof the eye. There was an infiltration of polymorphonuclear leukocytes(PMN) and lymphocytes in the vitreous space for the IRBP+PTX treatedmice (positive control for disease, no MSC) but no accompanyingdisorganization of retinal architecture, eg, retinal folds, nor damageto the photoreceptor layer (as comparing IRBP+PTX treated mice to MSConly treated mice, with the latter showing no uveitis disease, noinfiltrated immune cells). IRBP+PTX+MSC-treated mice showed a strikingreduction in the number of PMN and lymphocytes in the vitreous space (0or 1 cells) as compared to IRBP+PTX only mice (typically more than 40infiltrated immune cells in the vitreous space with additional cells inthe retinal architecture).

To further quantify the effect of MSC treatment, histological samplesand fundus examinations were independently scored in a blinded fashionby two investigators; the fundus and histological samples were performedin separate evaluations. The more severe the inflammation, inflammatorycell infiltration, and tissue damage, the higher the histology score.For funduscope examinations, higher scores represent more severe diseasefeatures. Additional disease features that do not readily appear inhistology, such as mild vasculitis and optic nerve head erythema can becaptured in funduscope imaging. Both eyes were scored separately foreach mouse. Raw scoring numbers per eye per mouse showed generalconsistency between histology and funduscope scores for each eye/mouse.The results further confirmed that administration of MSC decreased theseverity of uveitis in this model. Among the group A animals (in whichEAU was induced without MSC administration), histology scores were “0”in 5 eyes and “0.5” in 7 eyes, and funduscope scores were “0” in 1 eye,“0.5” in 4 eyes, “1” in 1 eye, “1.5” in 2 eyes, and “2” in 4 eyes. Amongthe group B animals (in which MSC were administered without EAUinduction), histology scores were “0” in all 16 eyes and funduscopescores were “0” in 11 eyes, “0.5” in 4 eyes, and “1” in 1 eye. Among thegroup C animals (in which EAU was induced and the MSC wereadministered), histology scores were “0” in 11 eyes, “0.25” in 2 eyes,and “0.5” in 1 eye; funduscope scores were “0” in 6 eyes, “0.5” in 3eyes, “1” in 3 eyes, and “1.5” in 2 eyes.

Example 22

This example describes performance of human ES cell-derived MSCs(hES-MSC) in an EAE model of multiple sclerosis. Particularly, it isdemonstrated in this example that hES-MSC outperform bone marrow MSC(BM-MSC) in this model. Additionally, the hES-MSC exhibited lower IL-6expression compared to BM-MSC, which may contribute to betterimmunosuppression. Additionally, hES-MSC exhibited superiorextravasation/migratory properties into injured CNS compared to BM-MSC.

Methods

Culture of hESCs and Generation of hES-MSCs

hESC lines were cultured either on Matrigel in TeSR1 medium or on MEFsin DMEM:F12+20% KOSR+10 ng/ml bFGF. To generate hES-MSCs, hESCs werefirst differentiated into EBs for 4 days and then hemangioblasts (HBs)for an additional 6-12 days as previously described (Lu et al., 2008).HB-containing cell clusters were then transferred to αMEM with 20% FBSand cultured on Matrigel (BD Bioscience)-coated plates at a density of5,000 cells/cm². The medium was refreshed every 3-4 days and after 7-14days, the adherent cells gradually differentiated into spindle-shapedcells.

Culture of BM-MSCs

BM-MSCs #1, 2, 3 were derived from BM mononuclear cells (BMMNCs)obtained from AllCells, Inc (Alameda); #4 was derived from Lonza (Basel,Switzerland) BMMNCs, while #5 (GFP-labeled) and #6 were obtained asBM-MSCs from Dr. Darwin Prockop at Texas A&M. For derivation of #1-4,BMMNCs were thawed and plated onto tissue culture plastic dishes inαMEM+20% FBS. Adherent cells began to appear within the first 4-5 daysand fed every 3 days until day ˜10-12, when cells were harvested andreplated at 3,000-5,000 cells/cm².

Analysis of Th1 and Th17 Cells in the CNS

EAE mice were perfused with cold PBS through the left ventricle. Brainand spinal cord were harvested, ground and digested with collagenase andDispase (1 mg/ml each) at 37° C. for 20 minutes. Single cells passedthrough 40 μm cell strainer were washed and re-suspended in 4 ml of 40%Percoll, overlaid onto 5 ml of 70% Percoll and centrifuged at 2,000 rpmfor 20 minutes. Inter layer cells were collected and stimulated with12-O-tetradecanoylphorbol-13-acetate (TPA) at 50 ng/ml (Sigma) andionomycin at 500 ng/ml (Sigma) in the presence of GolgiStop (BDBioscience) for 6 h. Cells were then immunostained with anti-CD4-FITCand anti-CD8-Pacific Blue (BD Bioscience). Flow cytometry for IFNγ andIL-17 was performed using an intracellular staining kit (BD Bioscience)according to manufacturer's instructions.

Pathology of the Spinal Cord

Paraffin-embedded spinal cord cross sections were immunostained for CD3(Biocare Medical) or Iba1 (Waco) and counterstained with anti-MBP(Millipore) or fluoromyelin as previously described (Crocker et al.,2006: Moore et al., 2011). Quantification of anti-MBP staining intensitywas performed on the lateral columns of sections from the thoracic andlumbar levels of spinal cord samples using Image) (NIH) (Crocker et al.,2006). At least three regions of interest were analyzed for each subjectin each treatment group.

In Vitro Assays of Th0, Th1, and Th17 Cells from Mouse Spleen

Naive CD4⁺ T cells were purified from mouse spleens with the Naive CD4 TCell Enrichment kit (Stem Cell Technologies) and activated with anti-CD3(1 μg/ml) and anti-CD28 (1 μg/ml). Cytokines and antibodies were addedas follow: Th0 condition, anti-mIFNγ (5 μg/ml) and anti-mIL-4 (5 μg/ml)(eBioscience); Th1 condition, rmIL-12 (10 ng/ml) (PeproTech), anti-mIL4(5 ug/ml); Th17 condition, anti-mIL-4 (5 μg/ml), anti-mIFNγ (5 μg/ml),rmIL-6 (20 ng/ml) (Peprotech), rhTGF-β 1 (1 ng/ml) (Peprotech). For somegroups, anti-hIL-6 (10 μg/ml) (eBioscience) was added. hES- or BM-MSCsthat had been mitotically-arrested through irradiation at 80 Gy, wereadded 1 hour later.

Cytokine Antibody Array

180,000 MSCs were plated per well of a 6-well plate and conditionedmedium (CM) was collected 3 days after plating. CM was spun down brieflyto remove cellular debris and then frozen at −20° C. CM was thawed foranalysis on RayBiotech (Norcross) custom membrane arrays according tomanufacturer's protocols.

Tracking of GFP⁺ MSCs in Perivascular Regions in the CNS

Spinal cord tissue was prepared as reported previously by Paul et al.(2013). In brief, after perfusion/fixation, spinal cords were harvestedby laminectomy, and cryosectioned for immunostaining. Anti-GFP-Alexa®488, anti-CD31 (BD Bioscience), Alexa® 555 secondary antibody (LifeTechnologies) were used to detect GFP⁺ and endothelial cells, DRAQ5(Biostatus Ltd.) were used to visualize the nuclei. Following staining,sections were mounted in Mowiol® and confocal z-stacks were acquired at1 μm increments between z-slices, following a multitrack scan, using aZeiss LSM 510 Meta confocal microscope. Images were analyzed withImaris® suite version 7.1 software (Bitplane Inc.). The GFP channel wasisosurface rendered to provide a better spatial perspective forvisualizing GFP⁺ cells.

Statistical Analysis of In Vivo Data

Clinical disease scores for each treatment group are expressed asmean±SE. The difference in 30 day clinical scores between groups wasanalyzed by two-way ANOVA and by t-test for the one-day scores incomparing the ability of GFP⁺hES-MSCs and GFP⁺BM-MSCs to extravasatefrom inflamed CNS venules and migrate into CNS parenchyma of EAE mice.Percentage data for CNS infiltrated T cells were aresine-transformedprior to analysis. SPSS 15 software was used for statistical analysis. Aprobability of P<0.05 was considered to be significant, and P<0.01 to behighly significant.

Characterization of hES-MSCs

Flow cytometry staining was performed using standard methods. Antibodiesinclude anti-CD31, CD34, CD29, CD73, CD90, CD105, CD44, CD45, CD166,HLA-ABC, HLA-DR, HLA-G (BD Bioscience, Biolegend, or eBioscience). Datawere collected on FAGS LSR 11 Flow Cytometer or Accuri C3 flowcytometer. Data analysis was performed with FlowJo (Treestar) or AccuriC6 software.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde, permeabilized with PBS+0.2%Triton X 100 and stained with primary antibody in 5% goat serumovernight at 4° C. Cells were then washed 3× with PBS, stained withfluorochrome-conjugated secondary antibodies, washed 3× with PBS, andmounted under cover slips prior to phase and fluorescence microscopy.

Multi-Lineage Differentiation of hES-MSCs

Chemicals for all 3 differentiation conditions were purchased fromSigma-Aldrich unless otherwise indicated. Undifferentiated MSCs werestained as controls. Osteogenic differentiation was performed aspreviously described (Karlsson et al., 2009), In brief, MSCs were grownin low glucose DMEM plus 10% FCS, 80 μM ascorbic acid 2-phosphate, 1 μMdexamethasone, and 20 mM beta-glycerophosphate, fed every 3-4 days for28 days, and stained with Alizarin Red. Adipogenic differentiation wasperformed as previously described (Karlsson et al., 2009). In brief,MSCs were grown in low glucose DMEM plus 20% FCS, 5 μg/ml insulin, 2 μMdexamethasone, 0.5 mM isobutylmethylxanthine, and 60 μM indomethacin,fed every 3-4 days for 30-32 days total, and stained with Oil Red O.Chondrogenic differentiation was performed as previously described(Barry et al., 2001). In brief, 2.5×10⁵ cells MSCs were pelleted in 15ml conical tubes and grown in high glucose DMEM, 1 mM Sodium Pyruvate,0.1 mM ascorbic acid 2-phosphate, 0.1 μM dexamethasone, 1% ITS(Collaborative Biomedical Products), and 10 ng/mL TGF-03 (Peprotech).Pellet cultures were fed every 2-3 days for 21-28 days total, fixed with4% paraformaldehyde, and sent to MassHistology (Worcester, Mass.) forparaffin embedding and sectioning prior to Alcian Blue and Nuclear FastRed staining.

Animal Model of Multiple Sclerosis

The mouse EAE model was induced as previously described (Stromnes andGoverman, 2006). In brief, C57BL/6 mice were subcutaneously injectedwith a mixture of MOG³⁵⁻⁵⁵, Freund's adjuvant, and pertussis toxincontained in the EAE Induction kit from Hooke Laboratories, Inc, MA(Cat. #EK-0114) following the manufacturer's protocol. BM- or hES-MSCsat 1×10⁶ cells/mouse or PBS (a vehicle control) were i.p. injected onday 6 (for pre-onset) or 18 (for post-onset) after the immunization.Disease score was monitored every day for up to 31 days as follows: 0,no sip of disease; 1, loss of tone in the tail; 2, partial hind limbparalysis; 3, complete hind limb paralysis; 4, front limb paralysis; and5, moribund (Stromnes and Goverman, 2006). For some experiments,cumulative and maximal disease scores were also calculated (e.g., inevaluating the ability of hES-MSCs to attenuate cumulative and maximaldisease scores in EAE mice).

In Vitro Assay of T Cell Proliferation

Lymphocytes isolated from mouse peripheral lymph nodes were labeled with5 μM of CFSE for 10 min. at 37° C. 1,000 hES- or BM-MSCs were mixed with10,000 lymphocytes per well in a 96-well plate, and the cells werestimulated for proliferation with plate-bound anti-CD3 and solubleanti-CD28 antibodies (eBioscience, CA). The cells were collected 3 daysafter the stimulation, followed by flow cytometry staining with anti-CD4and anti-CD8 antibodies (BD Bioscience, CA). CFSE dilution was gated onCD4⁺ and CD8⁺ T cells, respectively.

Quantitative RT-PCR

MSC RNA was isolated using the RNAeasy kit (Qiagen) per manufacturer'sinstructions. cDNA synthesis was performed using the SuperScript IIIFirst-Strand Synthesis SuperMix for qRT-PCR (Life Technologies).Quantitative RT-PCR (qRT-PCR) was performed using Taqman probes (LifeTechnologies) and SSO Fast Mastermix (Biorad) on a Biorad CFX 96 qRT-PCRmachine. Data was analyzed using CFX Manager 2.1 software (Biorad) andthe ΔΔCt method following normalization with GAPDH. For IL-6 RNAexpression in the stimulation experiments, MSCs were co-cultured withnon-adherent human PBMCs (containing T cells) for 4 days total. 2.5ng/ml PHA was added for the final 2 days. Non-adherent PBMCs were thenaspirated away and adherent cells were rinsed 3× with PBS prior to RNAprocessing.

Karyotyping: The G-banded karyotyping of hES-MSCs was conducted byCellLine Genetics (Madison, Wis.).

Human T Cell Proliferation Assayed Via Mixed Leukocyte Reaction to PHAStimulation

Non-adherent human PBMC from 2 different donors were used as a source ofT cell responders, hES-MSCs from 5 different lots and BM-MSCs from 3different donors were used as inhibitors among 4 independentexperiments. MSCs were mitotically-arrested with 5-10 μg/ml mitomycin C(Sigma-Aldrich) for 2.5-3 h, rinsed 2× with PBS and responder cells wereadded to the MSC culture at a 1:6 ratio in IMDM+10% heat-inactivatedhuman serum. On day 3, 2.5 ng/ml PHA (Sigma) was added to induce T cellproliferation. On day 4, 10 μM BrdU (BD Bioscience) was added and on day5, T cell proliferation was assessed through flow cytometric stainingusing anti-CD4-FITC, CD8-APC, and BrdU-PE antibodies with the BrdUIncorporation kit (BD Bioscience) according to manufacturer's protocol.

Human T_(reg) Cell Stimulation

Human PBMCs (AllCells) were co-cultured for 4 days in suspension withadherent hES-MSCs or BM-MSCs that had been previously mitoticallyinactivated with mitomycin C (Sigma-Aldrich). Media was IMDM (Gibco)+10%heat-inactivated human serum (Mediatech), and 30 IU/ml rhIL-2(Peprotech). T_(reg) cells were determined by triple positivity for CD4,CD25, and FoxP3 by staining with anti-CD4-FITC, CD25-APC, and FoxP3-PEantibodies using the Fox P3 Intracellular Flow Staining kit (Biolegend)according to manufacturer's protocol.

Results

MSCs are Efficiently Derived from hESCs Using Our HB-Enriching Method

Starting from either feeder-free hESCs or hESCs grown on mouse embryonicfibroblasts (MEFs), we obtained MSCs by using a 3-step approach thatgoes through both embryoid body (EB) and HB intermediate stages (FIG.33A). The HB intermediate contains a heterogeneous population of cellsincluding ˜68% CD45+ cells (hematopoietic progenitors) and ˜22% CD31+cells (endothelial progenitors) (FIG. 33B). Replating these cells onMatrigel in an αMEM-based MSC medium allowed the emergence of MSC-likecells; 24 h after plating, 5-10% of the cells attached to the plate and7-14 days thereafter, the attached cells fully differentiated intoMSC-like cells with cell surface markers similar to those of BM-MSCs.They expressed high levels of CD73 (>99%), CD90 (>90%), CD105 (>90%),CD13 (>85%), CD29 (>90%), CD54 (>80%), CD44 (>99%), and CD166 (>90%),but did not express non-MSC markers such as CD31, CD34 and CD45 (FIG.33B). Demonstrating the capability and reproducibility of this method,we found that cells harvested anytime between HB-d7 and HB-d12 couldsimilarly give rise to hES-MSCs. Specifically, flow cytometric analysisof cell surface markers on hESC-derived HB-enriched cells and hES-MSCswas performed and expression levels were determined from the mean offour independent experiments. hESC (MA09) derived-HB-enriched cells wereharvested after culture in methylcellulose-containing medium for 7-12days, and hES-MSCs were harvested after culture in the MSC Medium for14-21 days. In hES-MSC, over about 90-95% of cells were positive forCD105, CD90, CD29, CD73, CD166, CD44, and HLA-ABC, while less than 5% ofcells were positive for CD34, CD45, or CD31. In contrast, inhESC-HB-enriched cells, less than about 10% of cells were positive forCD105, CD90, CD73, CD34, and CD31; about 45% of cells were positive forCD29; about 30% of cells were positive for CD166, about 90% of cellswere positive for CD44; about 50% of cells were positive for HLA-ABC;and about 80% of cells were positive for CD45. Moreover, we have usedthis method to generate MSCs from 4 different hESC lines: H9 (Thomson etal., 1998), CT2 (derived at UConn)(Wang et al., 2009), MA09 (aFDA-approved, clinical-grade line derived at Advanced Cell Technology)(Klimanskaya et al., 2006), and ES03-Envy (Envy, a GFP-labeled linederived at ES International) (Costa et al., 2005).

Like BM-MSCs (Le Blanc et al., 2003; Selmani et al., 2008), our hES-MSCsexpress high levels of immunosuppressive non-classical MHC antigen HLA-G(FIG. 33C) and MHC class 1 antigen HLA-ABC (determined by flow cytometryas discussed above) but not MHC class II antigen HLA-DR andco-stimulatory molecule CD80; IFNγ treatment induces up-regulation ofHLA-DR but not CD80 (FIG. 33D). Functionally, the hES-MSCs aremultipotent as they can differentiate into osteocytes, adipocytes, andchondrocytes, a defining criterion for MSCs (FIG. 33E). The hES-MSCs canbe cultured in vitro for at least 12 passages and were determined tohave a normal karyotype at this time point; the cells also sustainedtheir immunophenotype and differentiation capabilities (data not shown).

hES-MSCs Attenuate the Disease Score of EAE Mice in Both Prophylacticand Therapeutic Modes

We employed a standard EAE model of MS in C57BL/6 mice to test thetherapeutic utility of our hES-MSCs. EAE was induced as previouslydescribed (Stromnes and Goverman, 2006). Six days after immunization,mice were injected with 1×10⁶ hES-MSCs or PBS intraperitoneally (i.p.).hES-MSCs derived from three hESC lines C72, MA09, and H9 allsignificantly attenuated the daily disease scores (FIG. 34A) as well ascumulative and maximal disease scores. However, mice injected withparental CT2 hESCs manifested high disease scores similar to those ofPBS controls (FIG. 34A). To confirm the clinical score data, we alsohistologically analyzed microglial inflammatory activity within thespinal cord. Immunostaining for ionized calcium-binding adapter molecule1 (IBA1) revealed reduced microgliosis in EAE mice treated with hES-MSCscompared to those treated with PBS (FIG. 34B). Infiltration of total Tcells (stained as CD3⁺ cells) into the spinal cord was also decreased byhES-MSC-treatment (FIG. 34B). Strong immunostaining for myelin-bindingprotein (MBP) indicates that demyelination was prevented in mice treatedwith hES-MSCs compared to those treated with PBS (FIGS. 34B and 34C).

In addition to the prophylactic effects shown above, we also tested theeffect of hES-MSCs treatment on mice that had already developed EAE(post-disease onset). When hES-MSCs were injected on day 18post-immunization, we observed a gradual decline in disease scores from˜3.0 down to an average score of 1.67 by day 30 in hES-MSCs-treatedmice, whereas the PBS-treated EAE mice showed an average score of 2.8 byday 30 (FIG. 34D). Collectively, data presented in FIG. 34 show thathES-MSCs can reproducibly decrease disease severity bothprophylactically and therapeutically in the EAE model.

Additionally, control experiments demonstrated that hES-MSCs, but notparental hESCs, attenuated cumulative and maximal disease scores in EAEmice. Cumulative and maximal disease scores during the course of 28-32days were compared for mice administered hES cells (CT2), hES-MSC (CT2),hES-MSC (H9), and hES-MSC (MA09), as well as negative controls(administered PBS only). N=5. Cumulative and maximal disease scores weresimilar for hES (CT2) administered mice and negative controls.Cumulative and maximal disease scores were significantly lower for eachgroup of mice that were administered hES-MSC compared to negativecontrols (P<0.01).

hES-MSCs Maintain Anti-EAE Properties and In Vivo Lifespan FollowingIrradiation

Some reports suggest that MSCs transplanted into animals may undergomalignant transformation or support tumor growth formed by host cells(Djouad et al., 2003; Wong, 2011). Yet, since short-term cytokinesecretion and cell-cell contact may be sufficient to exert MSC functions(Uccelli and Prockop, 2010), we hypothesized that irradiated MSCs maystill provide an anti-EAE effect. To test this hypothesis, we irradiatedhES-MSCs at 80 Gy immediately before injecting cells into EAE mice atday 6 post-immunization. We observed comparable anti-EAE effects to thenon-irradiated hES-MSC groups when we used 2×10⁶ cells/mouse ofirradiated cells (FIG. 35A). To test the actual lifespan of injectedcells, we transduced CT2 hESCs with the lentiviral luciferase-expressingvector (Pomper et al., 2009), and generated a stable luciferase-positivehESC-MSC line. Stable luciferase expression was confirmed byimmunostaining (not shown). We found that both the irradiated andnon-irradiated hES-MSCs had roughly the same lifespan of ˜7-10 days inthe mice (FIG. 35B).

Teratogenicity is an additional concern for any therapeutic cell typedifferentiated from pluripotent cells. To assess this risk, we injectedhES-MSCs into immunodeficient SCID-beige mice at 1×10⁶ cells/mouse, andfound no tumor formation at the injection sites within 2 months, whereasteratomas formed in mice injected with the same dose of parental hESCs(data not shown).

hES-MSCs have Stronger Anti-EAE Effects In Vivo than BM-MSCs

Given the reported variability in in vivo efficacy of BM-MSCs, wedecided to compare hES-MSCs and human BM-MSCs in the prophylactic EAEmodel. We obtained MSCs from 6 different BM donors and found 5 out ofthe 6 failed to significantly attenuate the disease score of EAE mice(FIGS. 36A, 36B, and GFP-expressing BM-MSCs) while one BM-MSC line(BM-MSC #6) showed a moderate effect (FIG. 36C). This is in markedcontrast to the four different hES-MSC lines that all showed a stronganti-EAE effect (FIG. 34A, and GFP-expressing hES-MSCs).

EAE/MS is accompanied by infiltration of reactive T cells into the CNS(McFarland and Martin, 2007). Therefore, we purified all T cells thathad penetrated into the CNS and found that hES-MSC-injected mice hadsignificantly fewer CD4+ and CD8+ (FIG. 36D) T cell infiltrates than PBScontrol EAE mice, coinciding with their reduction of disease severity.In contrast, BM-MSC-treated mice had the opposite effect and actuallydisplayed significantly more CD4+ and slightly more CD8+ T cellinfiltrates (FIG. 36D) than PBS control EAE mice. The percentage oftotal numbers of CD4+ and CD8+ cells in the CNS of EAE mice treated withPBS, hESC or hES-MSCs was determined on day 15 post-immunization.Lymphocytes purified from the CNS were analyzed for numbers of CD4+ andCD8+ cells by flow cytometry (N=4). The number of Th17-expressing cellswas significantly reduced (P<0.05) and the number of CD4, CD8, andTh1-expressing cells was highly significantly reduced (P<0.01) in theCNS of hES-MSC treated animals compared to PBS-treated controls.Parental hESC line CT2-injected mice had similar levels of CD4 and CD8cell infiltrates as PBS controls. We also found that hES-MSC-treated EAEmice had significantly fewer proinflammatory Th1 and Th17 cells thanPBS- or CT2 hESC-injected controls (FIG. 36D). Th1 (IFNγ⁺) and Th17(IL-17⁺) cells in the CNS of EAE mice treated with PBS, hES-MSCs (CT2)on day 15 post-immunization were also quantified by intracellular flowcytometry dot plots; in PBS-treated mice 11.49% of cells were IL-17⁺only, 15.16% of cells were IFNγ only, and 9.27% of cells were positivefor both IL-17 and IFNγ. In contrast, from hES-MSC treated mice 4.37% ofcells were IL-17⁺ only, 2.52% of the cells were IFNγ⁺ only, and 1.38% ofcells were positive for both IL-17 and IFNγ. BM-MSC-treated mice, on theother hand, had similar or greater Th1 numbers and consistently greaterTh17 numbers as compared to controls (FIG. 36D). Reduced fluoromyelinstaining of MBP in the spinal cord confirms severe damage in bothPBS-treated and BM-MSC-treated mice while MBP levels were preserved inhES-MSC-treated mice (FIG. 36E). The damaged regions in BM-MSC-treatedmice also show a high number of DAPI positive cells, suggesting moreinflammatory cells infiltration.

Considering the important role of regulatory T cells (T_(reg) cellsdetected as CD4+, Foxp3⁺, and CD25⁺) in suppressing inflammation, weexamined the ratio of T_(reg) cells among infiltrated CD4 T cells in theCNS and found no difference in hES-MSC-treated vs control EAE mice.Influence of hES-MSCs or BM-MSCs on regulatory T cell populations invivo and in vitro was examined by flow cytometry, and percentage ofFoxp3⁺/CD25⁺ cells among infiltrated CD4⁺ T cells in the CNS of EAE micetreated with hES-MSCs (CT2) or PBS was determined. In PBS treatedanimals, 5.17% of cells were Foxp3⁺ only, 11.5% of cells were CD25⁺only, and 21.7% of cells were both Foxp3⁺ and CD25⁺. In hES-MSC treatedanimals, 1.52% of cells were Foxp3⁺ only, 13.1% of cells were CD25⁺only, and 17.2% of cells were both Foxp3⁺ and CD25⁺. This is similar toa previous report on murine BM-MSCs in EAE (Zappia et al., 2005). Invitro, both hES- and BM-MSCs increased T_(reg) cell proliferation, butthere was no remarkable difference between the two groups. Human PBMCwere co-cultured with mitotically inactivated hES-MSCs (MA09) (n=6) orBM-MSCs (n=5) at a ratio of 1:10 and IL-2 at 30 IU/ml for 4 days.Percentages of T_(reg) cells were determined via flow cytometry stainingfor CD4⁺/CD25⁺/Foxp3⁺ cells and were less than 0.5% for T cells withoutIL-2, about 1.5% for T cells cultured with IL-2, about 5% for T cellscultured with hES-MSC and IL-2 (p<0.01 compared to T cells alone withIL-2), and about 4% for T cells cultured with BM-MSC and IL-2 (p<0.05compared to T cells alone with IL-2). This suggests that enhancedT_(reg) cell proliferation is a common response to both hES- andBM-MSCs, but not necessarily a contributor to the anti-EAE effect invivo.

hES-MSCs have Stronger Inhibition of T Cell Functions In Vitro thanBM-MSCs

We next compared hES-MSCs and BM-MSCs for their ability to inhibit Tcell proliferation in vitro. We stimulated carboxyfluoresceinsuccinimidyl ester (CFSE)-labeled mouse naïve T cells with increasingamounts of anti-CD3 antibody and found that hES-MSCs inhibited theproliferation of mouse CD4⁺ and CD8⁺ T cells when stimulated withanti-CD3 antibody at 0.2 and 0.6 μg/ml, or 0.1 and 0.3 μg/ml, whereasBM-MSCs only did so at low doses, i.e., 0.2 or 0.1 μg/ml of anti-CD3antibody. hES-MSCs were observed to have stronger suppression of T cellfunctions in vitro than BM-MSCs. hES (MA09)- or BM-MSCs were incubatedwith CFSE-labeled mouse lymphocytes at a ratio of 1:10 and stimulated byvarious concentrations of anti-CD3 antibody. The ratios of proliferatingCD4+ and CD8+ T cell were analyzed by flow cytometry (N=3). Flowcytometry histogram plots were carried out to show the percentage ofdivided CD4+ or CD8+ T cells with diluted CFSE signal.

Similarly, hES-MSCs showed stronger inhibition than BM-MSCs onproliferation of human T cells among phytohaemagglutinin(PHA)-stimulated human peripheral blood mononucleocytes (PBMCs).Mitotically inactivated hES (MA09)- or BM-MSCs were incubated withPHA-stimulated human PBMCs at a ratio of 1:6. The percentage ofproliferating CD4+ or CD8+ cells was measured with BrdU incorporationand the mean of 4 independent experiments was determined. The percentageof BrdU+ human T cells was approximately 0% for T cells only, 30% for Tcells only plus PHA, about 15% for T cells plus hES-MSC plus PHA, andabout 25% for T cells plus BM-MSC plus PHA. The difference in percentageof BrdU positive T cells was statistically significant between T cellsincubated with hES-MSC or BM-MSC (*P<0.05). Together, these data suggestthat hES-MSCs are more potent inhibitors of mouse and human T cellproliferation than BM-MSCs.

Since EAE mice treated with BM-MSCs had more Th1 and Th17 cellinfiltration in the CNS than mice treated with hES-MSCs (FIG. 36D), weexamined these T cell subtypes in vitro in the presence or absence ofhES- and BM-MSCs. Under the Th1 condition, differentiation of naïve CD4+T cells into Th1 (CD4⁺/IFNγ⁺) cells was reduced by hES-MSCs, but wasunaffected or even enhanced by different BM-MSC lines. In theseexperiments, hES (MA09)- or BM-MSCs were incubated with mouse naïve CD4⁺T cells at a ratio of 1:10, followed by Th1 or Th17 differentiation for5 days. IFNγ⁺ and IL-17⁺ CD4 T cells were detected via intracellularflow cytometry staining after TPA/ionomycin stimulation and results wereobtained from four independent experiments. Under Th1 condition, thefraction of CD4/IFNγ double positive cells was 29.7% in controls, 18.0%for hES-MSC, 26.9% for BM-MSC #2, 43.6% for BM-MSC #3, and 33.6% forBM-MSC #6. Of note, even the BM-MSC line that gave a modest therapeuticresponse in the EAE model (BM-MSC #6, FIG. 36C) could not effectivelyreduce Th1 differentiation in this in vitro assay. Under the Th17differentiation condition, both hES- and BM-MSCs reduced thedifferentiation of Th17 (CD4/IL17⁺) cells; the fraction of IFNγ⁻/IL17⁺cells was 21.4%, 5.15%, 1.13%, 2.73%, and 2.20% for control, hES-MSC,BM-MSC #2, BM-MSC #3, and BM-MSC #6, respectively. However, under thesame Th17-inducing conditions, BM-MSCs but not hES-MSCs significantlyincreased the percentage of IFNγ⁺/IL17⁻ (i.e., Th1) cells; the fractionof IFNγ⁺/IL17⁻ cells was 0.190%, 0.611%, 17.7%, 12.1%, and 14.2% forcontrol, hES-MSC, BM-MSC #2, BM-MSC #3, and BM-MSC #6, respectively.Collectively, these results show that hES-MSCs effectively dampen bothTh1 and Th17 differentiation cascades in vitro while BM-MSCs cansurprisingly promote Th1 differentiation under a Th17-inducingenvironment.

BM-MSCs Express Higher IL-6 than hES-MSCs, and IL-6 Blockage Enhancesthe Immunosuppressive Effect of BM-MSCs

A large number of soluble or matrix factors have been reported tomediate the immunomodulatory and/or neuroprotective effects of MSCs(Uccelli and Prockop, 2010). Therefore, we conducted microarray analysisto identify differences in the expression of these factors betweenBM-MSCs and hES-MSCs. The overall expressional profiles of hES- andBM-MSCs samples were quite similar (data not shown), yet a small set ofgenes was expressed differentially. Among these, IL6 appeared to be muchmore highly expressed in BM-MSCs than in hES-MSCs. Multiple methodsincluding qRT-PCR, intracellular flow cytometry, and cytokine antibodyarrays confirmed this finding. By qRT-PCR the average of threeindependent experiments demonstrated that the relative normalizedexpression of IL-6 was about 0.05 for hES-MSC and about 1.0 for BM-MSC(p<0.05). Intracellular flow cytometry staining was conducted for IL-6in BM-MSCs from 3 different donors and 3 hES-MSCs from three hESC lines.The percentages of of IL-6⁻/IL10⁻, IL-6⁺/IL10⁻, IL-6⁻/IL10⁺, andIL-6⁺/IL10⁻ cells respectively were 0.12, 0.00, 50.3, and 49.5 forBM-MSC #1; 0.05, 0.00, 65.7, and 34.2 for BM-MSC #2, 0.074, 0.00, 60.5,and 39.4 for BM-MSC #6, 0.25, 0.00, 96.7, and 3.05 for hES-MSC #1, 2.07,0.00, 96.9, and 1.07 for hES-MSC #2, and 0.00, 0.00, 97.2, and 2.81 forhES-MSC #3. Cytokine antibody arrays were also conducted to show thelevel of IL6 and IL8 proteins in conditioned medium from hES-MSCs orBM-MSCs. Cytokine antibodies were present on the membrane in duplicate,and array controls were also included. In representative mages of atleast 5 independent experiments it was observed that the detected levelIL-6 was substantially lower in the hES-MSC compared to BM-MSC.

Upon IFNγ stimulation, the percentage of IL-6 expressing hES-MSCs didnot change, however the percentage of IL6-expressing cells BM-MSCsnearly doubled, as determined by intracellular flow cytometry whichshowed the percentage of IL6-expressing cells +/−IFNγ (10 ng/ml for 12h). We also tested MSC production of IL6 following co-culture withstimulated PBMCs if cells as the latter produces high levels of IFNγ andTNFα upon stimulation. After removing the PBMCs in suspension, muchhigher IL-6 expression was detected, via qRT-PCR in the adherent BM-MSCsthan in the adherent hES-MSCs. Specifically, the IL-6 level in BM-MSCs,but not hES-MSCs (MA09), was dramatically enhanced following co-culturewith non-adherent human PBMCs for 4 days. In these experiments, 2.5ng/ml PHA was added for the final 2 days. PBMCs were washed away, andqRT-PCR was used to determine IL-6 expression normalized to GAPDH.Relative normalized IL-6 gene expression for hES-MSC with PHA-stimulatedT cells was about 3, but was about 25 for BM-MSC treated in the samemanner. When incubated with unstimulated T cells, relative normalizedIL-6 gene expression was about 1 for hES-MSC and about 2 for BM-MSC.

Since IL-6 has been found to enhance T cell survival and differentiation(Dienz and Rincon, 2009), we sought to determine the effects ofMSC-secreted human IL6 on mouse T cell differentiation. Under Th0conditions (i.e., without mouse cytokines plus anti-mouse IFNγ andanti-mouse IL4), co-culture with three different BM-MSC samples,including BM-MSC #6, was found to enhance the percentage ofIFNγ-secreting CD4+ T cells (Th1 cells) while hES-MSCs had no effect;for Th0 condition plus IgGkappa, the percentage of CD4+/IFN was 3.22,3.57, 39.4, 41.6, and 36.8 in controls, hES-MSC, BM-MSC #2, BM-MSC #3,and BM-MSC #6 treated cells, respectively. An anti-human-IL-6neutralizing antibody reduced this BM-MSC effect by ˜23%-50% (FIG. 6E).Specifically, for TH0 condition plus anti-IL-6, the percentage ofCD44+/IFN was 2.89, 0.672, 26.7, 21.4, and 28.2 in controls, hES-MSC,BM-MSC #2, BM-MSC #3, and BM-MSC #6 treated cells, respectively. Thus,elevated IL-6 production may be at least partially responsible for thecompromised anti-EAE effects of BM-MSCs by promoting pro-inflammatorydifferentiation of T cells.

Both hES-MSCs and BM-MSCs Home to Spinal Cord but Only hES-MSCsSuccessfully Extravasate into the Inflamed Tissue

To determine if hES- and BM-MSCs home to the injured CNS and extravasatethrough the damaged BBB and/or BSCB, we tracked the migration of MSCsdifferentiated from the constitutively GFP-expressing hESC line “Envy”(Costa et al., 2005), and GFP-labeled human BM-MSCs (Hofstetter et al.,2002) in EAE mice. Cells were injected on day 6 after immunization, andspinal cords were analyzed 8 days later, a time point when clinicalscores for GFP⁺BM-MSCs-injected mice and PBS controls were ˜1.5-2.0 andscores for GFP⁺hES-MSCs were effectively 0 when comparing averageclinical scores on day of spinal cord harvest (day 14 post immunization,N=3 mice per group). As indicated by 3D reconstruction of confocalz-stacks from fluorescently-stained spinal cord cryosections, while bothBM- and hES-MSCs homed to the injured EAE spinal cord, the vascularassociations of the two type MSC lines were vastly different. Bothparenchymal and meningeal venules were analyzed, as leukocytes arethought to first exit from the latter vascular bed duringneuroinflammation (Bartholomaus et al., 2009; Kivisakk et al., 2003).The GFP⁺ hES-MSCs were clearly observed to migrate out from both vesselpopulations into the perivascular space. With regard to parenchymalvessels, hES-MSCs also extended well beyond the perivascularspace—migrating deeper into the parenchymal tissue. hES-MSCs alsoconclusively extravasated across meningeal venules into the subarachnoidspace surrounding the spinal parenchyma. In marked contrast, GFP⁺BM-MSCs appeared to remain closely associated with parenchymal vessels,as if trapped inside or not capable of effectively exiting from vesselsand migrating into the parenchyma. BM-MSCs also failed to show asextensive extravasation from meningeal vessels compared to hES-MSC.These observations pinpoint a key difference between hES- and BM-MSCs invivo functionality in the EAE model. For the 3D reconstructions, 3Dconfocal datasets from spinal cord cryosections (60 μm) of MSC-injectedEAE mice were used. Sections were stained with anti-GFP to track MSC,anti-CD31 to identify endothelial cells, and DRAQ5 to label nuclei.Clusters of GFP+ MSCs were observed in the parenchymal and meningealvenules. GFP/DRAQ5 and isolated GFP channels were observed. Isosurfacerendered GFP+ cells, highlighted within the selected region of interest(ROI) were detectable. DISCUSSION

In this study, we describe a novel, reproducible, and highly efficientmethod for generating hES-MSCs and show that hES-MSCs derived frommultiple hESC lines all significantly attenuate disease scores in amouse EAE model of MS. In stark contrast, human BM-MSCs displayed littleto no therapeutic activity in the EAE model and limitedimmunosuppressive effects in vitro as compared to hES-MSCs.Mechanistically, the superior anti-EAE effects of hES-MSCs may, in part,be related to their lower expression of IL6 and greater ability toextravasate the BBB/BSCB and migrate into inflamed CNS tissue than theirBM-MSCs counterparts.

In examining the anti-EAE effects of hES-MSCs, we observed thatprophylactic treatment of EAE mice was more effective in attenuatingdisease scores than therapeutic treatment. This is not surprising sinceprophylactic treatment may only require MSC immunosuppressive activitywhile therapeutic intervention may additionally require extensive neuralrepair and regeneration. hES-MSCs may also contribute toneuroregeneration (perhaps through recruitment of endogenousprogenitors). More effective therapeutic intervention may be possiblewith larger doses and/or repeated injections of cells. Of note,irradiated hES-MSCs were also effective in reducing EAE disease scoreand had the same lifespan in vivo as their non-irradiated counterparts.Mitotic inactivation (through irradiation or mitomycin C treatment) isroutinely used in mixed leukocyte reaction (MLR) assays and does notappear to greatly affect MSC cytokine secretion or immunomodulatoryactivities in these assays (Bocelli-Tyndall et al., 2007). Thus,irradiation of cells may have important clinical value since being ableto reduce the concern for tumorigenic potential may outweigh a slightreduction in potency.

The muted in vivo efficacy of BM-MSCs that we observed is consistentwith previous reports that showed only mild (Gordon et al., 2008; Zhanget al., 2005) or negligible (Payne et al., 2012) effects of humanBM-MSCs on EAE mice. Interestingly, BM-MSC #6, the one BM-MSC line inour studies that caused a modest reduction in EAE clinical scores wasinjected at passage 2 while the other BM-MSCs, which showed no effect,were used at passage 3 or 4. Despite being injected at later passages(p4-6), all hES-MSC lines offered large reductions in the clinical scoreof EAE mice. This suggests that the therapeutic potency of BM-MSCs ismore sensitive to in vitro culture and expansion (a requirement forclinical scale use) than that of hES-MSCs. Indeed, other studies haveshown that BM-MSCs have lower proliferative capacity than do hES-MSCs(Sanchez et al., 2011) and may readily senesce with even limitedculturing (Wagner et al., 2008). While we did not observe massivesenescence or changes in immunophenotype in BM-MSC cultures by passage 4(data not shown), identifying biomarkers that accurately predict the invivo functionality of MSCs is an ongoing area of investigation in ourlab and others.

In our search for molecular differences between hES- and BM-MSCs, wefound that IL-6 was expressed much higher in BM-MSCs than in hES-MSCs inboth the basal and IFNγ-stimulated state. Elevated IL-6 levels have beenfound in blood and brain tissue from MS patients (Patanella et al.,2010) while site-specific production of IL-6 in the CNS can re-targetand enhance inflammation in EAE (Quintana et al., 2009). Interestingly,mice lacking IL-6 receptor a at the time of T cell priming are resistantto EAE (Leech et al., 2012) and an IL-6-neutralizing antibody can reducesymptoms in EAE mice (Gijbels et al., 1995). As discussed further below,high levels of IL-6 may contribute to functional differences betweenBM-MSCs and hES-MSCs in terms of how they influence immune effectorcells and how potent they are in a therapeutic setting. Indeed, it wouldbe interesting to determine if combined treatment of BM-MSCs with anIL-6-neutralizing antibody or use of BM-MSCs with IL-6 knockdownenhances the anti-EAE effect.

Many studies have reported that MSCs can inhibit the differentiation ofboth Th1 and Th17 cells yet recent reports have noted that they canactually promote differentiation of pro-inflammatory T cells undercertain permissive environments (Carrion et al., 2011; Darlington etal., 2010). In vivo and in comparison to PBS controls, we observedreduced CNS infiltration of Th1 and Th17 cells with hES-MSC treatmentbut increased CNS infiltration of Th1 and Th17 cells with BM-MSCtreatment. In vitro and unlike hES-MSCs, we found that BM-MSCs skewed Tcell differentiation to a Th1 phenotype under both Th0 (non-polarizing)and Th17 conditions. It is possible that certain factors produced highlyby human BM-MSCs, but not hES-MSC, can trigger Th1 differentiation, thusoverriding Th17 differentiation under these artificial in vitroconditions (Lazarevic et al., 2011). An anti-human IL-6 antibody wasable to partially reverse the effect under Th0 conditions but not Th17conditions (data not shown), presumably because the presence ofexogenous mouse IL-6, which is added for Th17-induction, cannot beneutralized by the anti-human IL-6 antibody. Together these suggest thathigh IL6 secretion by BM-MSCs may impact the local cytokine milieu andaugment the overall inflammatory response resulting into a strikingdifference in Th1/Th17 CNS infiltration between hES- and BM-MSC-treatedmice. Lastly, as the BBB and BSCB show evidence of structural damage andfunctional impairment in EAE (Bennett et al., 2010), it is possible thatMSCs at least partially execute their therapeutic effects in thisdisease by repairing these barriers (Pati et al., 2011). Since bothGFP-labeled hES- and BM-MSC homed to the CNS, while only hES-MSC showedhigh therapeutic potential with capacity to effectively extravasate andmigrate into the parenchyma, we hypothesize therapeutic efficacy andextravasation may be mechanistically linked. That MSCs might need toextravasate during EAE therapy is consistent with evidence these cellscan downregulate pro-inflammatory effector functions of parenchymalmicroglia (FIG. 34B) (Lee et al., 2012; Sheikh et al., 2011). It issignificant that in vitro culture and expansion of MSCs have both beenimplicated as factors that impair homing and transendothelial migration(De Becker et al., 2007; Rombouts and Ploemacher, 2003). A priori, itmay be that BM-MSCs are more sensitive to these factors than hES-MSCs,and this is a determinant of the differences in therapeutic efficacyobserved between the two MSC types. A variety of adhesion molecules andchemokine receptors, including α4β1, α4β7 integrins and several CXCRsand CCRs that are involved in leukocyte transmigration may alsoparticipate in MSC transmigration (Chamberlain et al., 2011). However,MSC extravasation may involve additional or unique mechanisms (Teo etal., 2012)—especially in the context of breaching the BBB and BSCB.Determining the dynamic changes and differences in expression of thesemolecules between hES- and BM-MSCs in situ will be critical indelineating the molecular requirements for MSC extravasation. In situgene profiling of both MSC types is currently in play, and shouldfurther shed light on the mechanism(s) responsible for the uniquetherapeutic efficacy of hES-MSCs.

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J Neuroimmunol 227, 185-189.-   Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I.,    Gerdoni, E., Giunti, D., Ceravolo, A., Cazzanti, F., Frassoni, F.,    et al. (2005). Mesenchymal stem cells ameliorate experimental    autoimmune encephalomyelitis inducing T-cell anergy. Blood 106,    1755-1761.-   Zhang, J., Li, Y., Chen, J., Cui, Y., Lu, M., Elias, S. B.,    Mitchell, J. B., Hammill, L., Vanguri, P., and Chopp, M. (2005).    Human bone marrow stromal cell treatment improves neurological    functional recovery in EAE mice. Exp Neurol 195, 16-26.

Example 23

Lupus Model: Administration of MSCs was Highly Effective in ProlongingSurvival of BWF1 Mice

(NZB×NZW)F1 (BWF1) lupus-prone mice were treated with hESC-derived MSCseither in one single dose of 2×10⁶ cells, or in two doses of 0.5×10⁶cells per dose, with second dose being administered two weeks after thefirst (day 0). The rate of survival of hESC-MSC treated mice wascompared to control animals. The hESC-treated and control animal cohortsincluded 20 animals each. As shown in FIG. 37A, forty percent (40%) ofthe control animals were deceased within 50 days, while the animalstreated with the hESC-derived MSCs had a substantially improved rate ofsurvival in comparison (95%-100% survival at 50 days). Relative to thecontrol group, the animals treated with hESC-derived MSCs also showed ahigher average weight after 7 weeks (post treatment), as well asincreases in CD4+ Foxp3+ T cells and Regulatory T cells in peripheralblood samples. As shown in FIG. 37B, mice treated with the hESC-derivedMSCs also had substantially lower levels of proteinuria (presence of anexcess of serum proteins in the urine) relative to the control group.

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1. A pharmaceutical preparation suitable for use in a mammalian patient,comprising at least 10⁶ mesenchymal stromal cells and a pharmaceuticallyacceptable carrier, wherein the mesenchymal stromal cells havereplicative capacity to undergoes at least 10 population doublings incell culture with less than 25 percent of the cells undergoing celldeath, senescing or differentiating into non-MSC cells by the tenthdoubling, or (b) at least 5 passages in cell culture with less than 25percent of the cells undergoing cell death, senescing or differentiatinginto fibroblasts by the fifth passage, wherein the mesenchymal stromalcells are obtained by in vitro differentiation of a hemangioblast cellinto a mesenchymal stromal cell.
 2. (canceled)
 3. A pharmaceuticalpreparation comprising at least 10⁶ mesenchymal stromal cells and apharmaceutically acceptable carrier, wherein the mesenchymal stromalcells are obtained by in vitro differentiation of a hemangioblast cellinto a mesenchymal stromal cell.
 4. A cryogenic cell bank comprising atleast 10⁸ mesenchymal stromal cells, wherein the mesenchymal stromalcells have replicative capacity to undergo at least 10 populationdoublings in cell culture with less than 25 percent of the cellsundergoing cell death, senescing or differentiating into fibroblasts bythe tenth population doubling, wherein the mesenchymal stromal cells areobtained by in vitro differentiation of a hemangioblast cell into amesenchymal stromal cell. 5.-103. (canceled)
 104. A kit comprising thepreparation of mesenchymal stromal cells of claim 1, wherein said cellsor preparation of cells are frozen or cryopreserved, or wherein saidcells or preparation of cells is contained in a cell delivery vehicle.105. (canceled)
 106. A method for treating a disease or disorder,comprising administering an effective amount of the preparation ofmesenchymal stromal cells according to claim 1 to a subject in needthereof. 107.-112. (canceled)
 113. A method of treating bone loss orcartilage damage comprising administering an effective amount of thepreparation of mesenchymal stromal cells according to claim 1 to asubject in need thereof. 114.-125. (canceled)
 126. The pharmaceuticalpreparation of claim 1, wherein basal CD24 expression is upregulated inmesenchymal stromal cells of the pharmaceutical preparation relative tomesenchymal stromal cells of bone marrow.
 127. The pharmaceuticalpreparation of claim 3, wherein basal CD24 expression is upregulated inmesenchymal stromal cells of the pharmaceutical preparation relative tomesenchymal stromal cells of bone marrow.
 128. The cryogenic cell bankof claim 4, wherein basal CD24 expression is upregulated in mesenchymalstromal cells of the cryogenic cell bank relative to mesenchymal stromalcells of bone marrow.